Method and compositions for treating cellulose containing fabrics using truncated cellulase enzyme compositions

ABSTRACT

Improved methods of treating cellulose containing fabrics with cellulase comprising contacting the cellulose fabrics with truncated cellulase enzyme. Treatment of cellulose containing fabrics with cellulase core domains of the invention are disclosed as offering specific advantages of reduced redeposition of dye and increased abrasion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/382,452, filed Feb. 1,1995 now U.S. Pat. No. 6,268,196 which is a continuation-in-part of U.S.Ser. No. 08/169,948 filed Dec. 17, 1993, now U.S. Pat. No. 5,861,271 nowpending and which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention is directed to improved methods for treatingcotton-containing fabrics and non-cotton containing cellulose fabricswith cellulase as well as to the fabrics produced from these methods. Inparticular, the improved methods of the present invention are directedto contacting cotton-containing fabrics and non-cotton containingfabrics with an aqueous solution containing a cellulase compositionwhich comprises one or more truncated cellulase enzymes.

B. State of the Art

During or shortly after their manufacture, cotton-containing fabrics canbe treated with cellulase in order to impart desirable properties to thefabric. For example, in the textile industry, cellulase has been used toimprove the feel and/or appearance of cotton-containing fabrics, toremove surface fibers from cotton-containing knits, for imparting astone washed appearance to cotton-containing denims and the like.

In particular, Japanese Patent Application Nos. 58-36217 and 58-54032 aswell as Ohishi et al., “Reformation of Cotton Fabric by Cellulase” andJTN December 1988 journal article “What's New—Weight Loss Treatment toSoften the Touch of Cotton Fabric” each disclose that treatment ofcotton-containing fabrics with cellulase results in an improved feel forthe fabric. It is generally believed that this cellulase treatmentremoves cotton fuzzing and/or surface fibers which reduces the weight ofthe fabric. The combination of these effects imparts improved feel tothe fabric.

Additionally, it was heretofore known in the art to treatcotton-containing knitted fabrics with a cellulase solution underagitation and cascading conditions, for example, by use of a jet, forthe purpose of removing broken fibers and threads common to theseknitted fabrics.

Clothing made from cellulose fabric, such as cotton denim, is stiff intexture due to the presence of sizing compositions used to easemanufacturing, handling and assembling of clothing items and typicallyhas a fresh dark dyed appearance. One desirable characteristic ofindigo-dyed denim cloth is the alteration of dyed threads with whitethreads, which gives denim a white on blue appearance.

After a period of extended wear and laundering, the clothing items,particularly denim, can develop in the clothing panels and on seams,localized areas of variation in the form of a lightening, in the depthor density of color. In addition, a general fading of the clothes, somepucker in seams and some wrinkling in the fabric panels can oftenappear. Additionally, after laundering, sizing is substantially removedfrom the fabric resulting in a softer feel. In recent years such adistressed or “stonewashed” look, particularly in denim clothing, hasbecome very desirable to a substantial proportion of the public.

Previous methods for producing the distressed look included stonewashingof a clothing item or items in a large tub with pumice stones having aparticle size of about 1 to 10 inches and with smaller pumice particlesgenerated by the abrasive nature of the process. Typically the clothingitem is tumbled with the pumice while wet for a sufficient period suchthat the pumice abrades the fabric to produce in the fabric panels,localized abraded areas of lighter color and similar lightened areas inthe seams. Additionally the pumice softens the fabric and produces afuzzy surface similar to that produced by the extended wear andlaundering of the fabric. This method produced the desired white on bluecontrast described above.

The use of the pumice stones has several disadvantages, includingoverload damage to the machine motors, mechanical damage to transportmechanisms and washing drums, environmental waste problems from the gritproduced and high labor costs associated with the manual removal of thestones from the pockets of the garments.

In view of the problems associated with pumice stones in stonewashing,cellulase solutions are used as a replacement for the pumice stonesunder agitating and cascading conditions, i.e., in a rotary drum washingmachine, to impart a “stonewashed” appearance to the denim (U.S. Pat.No. 4,832,864).

Cellulases are enzymes which hydrolyze cellulose (β-1,4-D-glucanlinkages) and produce as primary products glucose, cellobiose,cellooligosaccharides, and the like. Cellulases are produced by a numberof microorganisms and comprise several different enzyme classificationsincluding those identified as exo-cellobiohydrolases (CBH),endoglucanases (EG) and β-glucosidases (BG) (Schulein, M, 1988 Methodsin Enzymology 160: 235-242).

The enzymes within these classifications can be separated intoindividual components. For example, the cellulase produced by thefilamentous fungus, Trichoderma longibrachiatum, hereafter T.longibrachiatum, consists of at least two CBH components, i.e., CBHI andCBHII, and at least four EG components, i.e., EGI, EGII, EGIII and EGV(Saloheimo, A. et al 1993 in Proceedings of the second TRICEL symposiumon Trichoderma reesei Cellulases and Other Hydrolases, Espoo, Finland,ed by P. Suominen & T. Reinikainen. Foundation for Biotechnical andIndustrial Fermentation Research 8:139-146) components, and at least oneβ-glucosidase. The genes encoding these components are namely cbh1,cbh2, egl1, egl2, egl3, and egl5 respectively.

The complete cellulase system comprising CBH, EG and BG componentssynergistically act to convert crystalline cellulose to glucose. The twoexo-cellobiohyrolases and the four presently known endoglucanases acttogether to hydrolyze cellulose to small cello-oligosaccharides. Theoligosaccharides (mainly cellobioses) are subsequently hydrolyzed toglucose by a major β-glucosidase (with possible additional hydrolysisfrom minor β-glucosidase components).

A problem with the use of complete cellulase compositions fromTrichoderma sp. microorganisms and other fungal sources for stonewashingdyed denim is the incomplete removal of colorant caused by redepositionor backstaining of some of the dye back onto the cloth during thestonewashing process. In the case of denim fabric, this causesrecoloration of the blue threads and blue coloration of the whitethreads, resulting in less contrast between the blue and white threadsand abrasion points (i.e., a blue on blue look rather than the preferredwhite on blue). See, American Dyestuff Reporter, September 1990, pp.24-28. This redeposition is objectionable to some users.

Trichoderma cellulases, even though they result in backstaining arepreferred because of their higher activity on denim material. Inaddition, cellulases with a higher degree of purity may be beneficial inthe present invention. High specific activity or a high level of purityresults in a higher degree of abrasion in a significantly shorterprocessing time and therefor, is preferable to the denim processors.

Attempts to reduce the amount of redeposition of dye included theaddition of extra chemicals or enzymes, such as surfactants, proteasesor other agents, into the cellulase wash to help disperse the looseneddye. In addition, processors have used less active whole cellulase,along with extra washings. However, this results in additional chemicalcosts and longer processing times. Another method includes the use of amild bleach agent or stain removing agent in the process. This methodaffects the garment's final shade and increases the processing time.Finally the use of enzymes and stones together leave the processor withall the problems caused by the use of the stones alone. Accordingly, itwould be desirable to find a method to prevent redeposition of colorantduring stonewashing with cellulases.

Protein analysis of the cellobiohydrolases (CBHI and CBHII) and majorendoglucanases (EGI and EGII) of T. longibrachiatum has shown that abifunctional organization exists in the form of a catalytic core domainand a smaller cellulose binding domain separated by a linker or flexiblehinge stretch of amino acids rich in proline and hydroxyamino acids.Genes for the two cellobiohydrolases, CBHI and CBHII (Shoemaker, S et al1983 Bio/Technology 1, 691-696, Teeri, T et al 1983, Bio/Technology 1,696-699 and Teeri, T. et al, 1987, Gene 51, 43-52) and two majorendoglucansases, EGI and EGII (Penttila, M. et al 1986, Gene 45,253-263, Van Arsdell, J. N/et al 1987 Bio/Technology 5, 60-64 andSaloheimo, M. et al 1988, Gene 63, 11-21) has been isolated from T.longibrachiatum and the protein domain structure has been confirmed.

A similar bifunctional organization of cellulase enzymes is found inbacterial cellulases. The cellulose binding domain (CBD) and catalyticcore of Cellulomonas fimi endoglucanase A (C. fimi Cen A) has beenstudied extensively (Ong E. et al 1989, Trends Biotechnol. 7:239-243,Pilz et al 1990, Biochem J. 271:277-280 and Warren et al 1987, Proteins1:335-341). Gene fragments encoding the CBD and the CBD with the linkerhave been cloned, expressed in E. coli and shown to possess novelactivities on cellulose fibers (Gilkes, N. R. et al 1991, Microbiol.Rev. 55:305-315 and Din, N et al 1991, Bio/Technology 9:1096-1099). Forexample, isolated CBD from C. fimi Cen A genetically expressed in E.coli disrupts the structure of cellulose fibers and releases smallparticles but has no detectable hydrolytic activity. CBD further possessa wide application in protein purification and enzyme immobilization. Onthe other hand, the catalytic domain of C. fimi Cen A isolated fromprotease cleaved cellulase does not disrupt the fibril structure ofcellulose and instead smooths the surface of the fiber.

Trichoderma longibrachiatum CBHI core domains have been separatedproteolytically and purified but only milligram quantities are isolatedby this biochemical procedure (Offord D., et al 1991, Applied Biochem.and Biotech. 28/29:377-386). Similar studies were done in an analysis ofthe core and binding domains of CBHI, CBHII, EGI and EGII isolated fromT. longibrachiatum after biochemical proteolysis, however, only enoughprotein was recovered for structural and functional analysis (Tomme, Pet al, 1988, Eur.J. Biochem 170:575-581 and Ajo, S, 1991 FEBS291:45-49). Accordingly, the prior art has failed to recognize theimprovements possible in textile processing when using cellulase coredomain or cellulose binding domain regions.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of treatingcellulose containing fabrics which results in reduced redeposition ofdye at an equivalent level of abrasion of the fabric treated over priorart methods.

It is a further object of the present invention to provide a detergentcomposition having the properties of reduced redeposition of dye at anequivalent level of abrasion over prior art detergent compositions.

It is a further object of the present invention to provide astonewashing composition having the properties of reduced redepositionof dye at an equivalent level of abrasion over prior art stonewashingcompositions.

According to the present invention a method is provided for treatingcellulose containing fabrics with cellulase comprising the steps of: (a)contacting said cellulose containing fabric with a treating compositioncomprising an effective amount of truncated cellulase enzyme, derivativethereof or naturally occurring cellulase containing no cellulase bindingdomain; and (b) incubating said cellulose containing fabric in contactwith said truncated cellulase enzymes, derivative thereof or naturallyoccurring cellulase containing no cellulase binding domain for a timeand under temperature effective to treat said fabric. Also, acomposition is provided for treating a cellulose containing fabriccomprising a truncated cellulase. In a preferred embodiment the methodof treating comprises laundering or stonewashing. Also, preferably, thetruncated enzyme comprises a truncated cellulase core. Most preferablythe truncated cellulase core comprises EGI core, EGII core, CBHI core,or CBHII core. Further preferably, the cellulase is present in aconcentration of from about 0.1 to 1,000 ppm, more preferably from about0.5 to about 250 ppm.

According to a preferred embodiment of the present invention, adetergent or stonewashing composition is provided comprising truncatedcellulase enzyme. Because of the surprising reduced redepositionactivity of the truncated cellulase enzyme compositions according to thepresent invention, cellulose containing fabrics would be enhanced to asurprising extent upon cleaning or stonewashing with a suitablecomposition comprising truncated enzyme.

An advantage of the present invention is that a cellulase compositioncomprising truncated cellulase core enzymes, alone or in combinationwith other truncated cellulase core or non-truncated cellulases, isprovided which confers desirable qualities to cellulose containingfabrics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a)-1(c) depict the genomic DNA and amino acid sequence of CBHIderived from Trichoderma longibrachiatum. The signal sequence beings atbase pair 210 and ends at base pair 260 (Seq. ID No. 25). The catalyticcore domain begins at base pair 261 through base pair 671 of the firstexon, base pair 739 through base pair 1434 of the second exon, and basepair 1498 through base pair 1713 of the third exon (Seq ID No. 9). Thelinker sequence begins at base pair 1714 and ends at base pair 1785(Seq. ID No. 17). The cellulose binding domain begins at base pair 1786and ends at base pair 1888 (Seq ID No. 1). Seq ID Nos. 26, 10, 18 and 2represent the amino acid sequence of the CBHI signal sequence, catalyticcore domain, linker region and binding domain, respectively.

FIGS. 2(a)-2(e) depict the genomic DNA and amino acid sequence of CBHIIderived from Trichoderma longibrachiatum. The signal sequence begins atbase pair 614 and ends at base pair 685 (Seq ID No. 27). The cellulosebinding domain begins at base pair 686 through base pair 707 of exonone, and base pair 755 through base pair 851 of exon two (Seq ID No. 3).The linker sequence begins at base pair 852 and ends at base pair 980(Seq ID No. 19). The catalytic core begins at base pair 981 through basepair 1141 of exon two pair 1199 through base pair 1445 of exon three andbase pair 1536 through base pair 2221 of exon four (Seq ID No. 11). SeqID Nos. 28, 4, 20 and 12 represent the amino acid sequence of the CBHIIsignal sequence, binding domain, linker region and catalytic coredomain, respectively.

FIGS. 3(a)-3(c) depict the genomic DNA and amino acid sequence of EGI.The signal sequence begins at base pair 113 and ends at base pair 178(Seq ID No. 29). The catalytic core domain begins at base pair 179through 882 of exon one, and base pair 963 through base pair 1379 of thesecond exon (Seq ID No. 13). The linker region begins at base pair 1380and ends at base pair 1460 (Seq ID No. 21). The cellulose binding domainbegins at base pair 1461 and ends at base pair 1616 (Seq ID No. 5). Seq.ID Nos. 30, 14, 22 and 6 represent the amino acid sequence of EGI signalsequence, catalytic core domain, linker region and binding domain,respectively.

FIGS. 4(a)-4(c) depict the genomic DNA and amino acid sequence of EGII.The signal sequence begins at base pair 262 and ends at base pair 324(Seq ID No. 31). The cellulose binding domain begins at base pair 325and ends at base pair 432 (Seq ID No. 7). The linker region begins atbase pair 433 and ends at base pair 534 (Seq No. 23). The catalytic coredomain begins at base pair 535 through base pair 590 in exon one, andbase pair 765 through base pair 1689 in exon two (Seq ID No. 15). Seq IDNos. 32, 8, 24 and 16 represent the amino acid sequence of EGII signalsequence, binding domain, linker region and catalytic core domain,respectively.

FIGS. 5(a)-5(b) depict the genomic DNA and amino acid sequence of EGIII.The signal sequence begins at base pair 151 and ends at base pair 198(Seq ID No. 35). The catalytic core domain begins at base pair 199through base pair 557 in exon one, base pair 613 through base pair 833in exon two and base pair 900 through base pair 973 in exon three (SeqID No. 33). Seq. ID Nos. 36 and 34 represent the amino acid sequence ofEGIII signal sequence and catalytic core domain, respectively.

FIGS. 6(a)-6(b) illustrate the construction of EGI core domainexpression vector (Seq ID No. 37).

FIGS. 7(a)-7(b) depict the construction of the expression plasmid pTEX(Seq ID Nos. 39-41).

FIG. 8 is an illustration of the construction of CBHI core domainexpression vector (Seq ID No. 38).

FIG. 9 is an illustration of the construction of the CBHII core domainexpression vector.

FIG. 10 illustrates abrasion/redeposition results obtained which compareEGI with EGI core and EGII with EGII core.

FIG. 11 illustrates the improvement in abrasion/redeposition results ofEGIII treated denim when CBHI core is added.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“Cotton-containing fabric” means sewn or unsewn fabrics made of purecotton or cotton blends including cotton woven fabrics, cotton knits,cotton denims, cotton yarns and the like. When cotton blends areemployed, the amount of cotton in the fabric should be at least about 40percent by weight cotton; preferably, more than about 60 percent byweight cotton; and most preferably, more than about 75 percent by weightcotton. When employed as blends, the companion material employed in thefabric can include one or more non-cotton fibers including syntheticfibers such as polyamide fibers (for example, nylon 6 and nylon 66),acrylic fibers (for example, polyacrylonitrile fibers), and polyesterfibers (for example, polyethylene terephthalate), polyvinyl alcoholfibers (for example, Vinylon), polyvinyl chloride fibers, polyvinylidenechloride fibers, polyurethane fibers, polyurea fibers and aramid fibers.

“Cellulose containing fabric” means any cotton or non-cotton containingcellulosic fabric or cotton or non-cotton containing cellulose blendincluding natural cellulosics and manmade cellulosics (such as jute,flax, ramie, rayon, TENCEL™). Included under the heading of manmadecellulose containing fabrics are regenerated fabrics that are well knownin the art such as rayon. Other manmade cellulose containing fabricsinclude chemically modified cellulose fibers (e.g, cellulose derivatizedby acetate) and solvent-spun cellulose fibers (e.g. lyocell). Of course,included within the definition of cellulose containing fabric is anygarment or yarn made of such materials. Similarly, “cellulose containingfabric” includes textile fibers made of such materials.

“Treating composition” means a composition comprising a truncatedcellulase component which may be used in treating a cellulose containingfabric. Such treating includes, but is not limited to, stonewashing,modifying the texture, feel and/or appearance of cellulose containingfabrics or other techniques used during manufacturing of cellulosecontaining fabrics. Additionally, treating within the context of thisinvention contemplates the removal of “dead cotton”, from cellolosicfabric or fibers, i.e. immature cotton which is significantly moreamorphous than mature cotton. Dead cotton is known to cause unevendyeing. Additionally, “treating composition” means a compositioncomprising a truncated cellulase component which may be used in washingof a soiled manufactured cellulose containing fabric. For example,truncated cellulase may be used in a detergent composition for washinglaundry. Detergent compositions useful in accordance with the presentinvention include special formulations such as pre-wash, pre-soak andhome-use color restoration compositions. Treating compositions may be inthe form of a concentrate which requires dilution or in the form of adilute solution or form which can be applied directly to the cellulosecontaining fabric.

It is Applicants' present belief that the action pattern of cellulaseupon cellulose containing fabrics does not differ significantly whetherused as a stonewashing composition during manufacturing or duringlaundering of a soiled manufactured cellulose containing fabric. Thus,improved properties such as abrasion, redeposition of dye, strength lossand improved feel conferred by a certain cellulase or mixture ofcellulases are obtained in both detergent and manufacturing processesincorporating cellulase. Of course, the formulations of specificcompositions for the various textile applications of cellulase, e.g.,stonewashing or laundry detergent or pre-soak, may differ due to thedifferent applications to which the respective compositions aredirected, as indicated herein. However, the improvements effected by theaddition of cellulase compositions will be generally consistent througheach of the various textile applications.

“Stonewashing composition” means a formulation for use in stonewashingcellulose containing fabrics. Stonewashing compositions are used tomodify cellulose containing fabrics prior to presentation for consumersale, i.e., during the manufacturing process, in contrast to detergentcompositions which are intended for the cleaning of soiled garments.

“Stonewashing” means the treatment of colored cellulose containingfabric with a cellulase solution under agitating and cascadingconditions, i.e., in a rotary drum washing machine, which impart a“stonewashed” appearance to the denim. Methods for imparting astonewashed appearance to denim are described in U.S. Pat. No. 4,832,864which is incorporated herein by reference in its entirety. Generally,stonewashing techniques have been applied to dyed denim.

“Detergent composition” means a mixture which is intended for use in awash medium for the laundering of soiled cellulose containing fabrics.In the context of the present invention, such compositions may include,in addition to cellulases and surfactants, many additives, including,but not limited to, additional hydrolytic enzymes, builders, bleachingagents, bluing agents and fluorescent dyes, caking inhibitors, maskingagents, cellulase activators, antioxidants, and solubilizers may beincluded. Such compositions are generally used for cleaning soiledgarments and are not used during the manufacturing process, in contrastto stonewashing compositions.

“Redepositing cellulase” means cellulases which in the enzymaticstonewashing or other treatment of cellulose containing fabrics usingcellulase solutions, particularly denim, result in redeposition of dyeonto the substrate. This effect is often referred to as backstaining.Such backstaining of the fabric leads to incomplete stonewashing becauseinstead of the desired blue on white contrast, the redeposition resultsin blue on blue. Redepositing cellulases include those derived frommicroorganisms such as the fungal microorganism Trichoderma sp. and thelike. In particular, EGI, EGII, CBHI and CBHII are known to exhibitsignificant redeposition behavior in their non-truncated state.

“Surface active agent or surfactant” means anionic, non-ionic andampholytic surfactants well known for their use in detergentcompositions.

“Wash medium” means an aqueous wash solution prepared by adding arequisite amount of a detergent composition to water. The wash mediumgenerally contains a cleaning effective amount of the detergent.

“Cellulolytic enzymes” or “Cellulase enzymes” means fungal exoglucanasesor exo-cellobiohydrolases, endoglucanases, and β-glucosidases. Thesethree different types of cellulase enzymes act synergistically toconvert cellulose and its derivatives to glucose. Analysis of the genescoding for CBHI, CBHII, EGI, EGII and EGV in Trichoderma longibrachiatumshows a domain structure comprising a catalytic core region or domain(CCD), a hinge or linker region (used interchangeably herein) andcellulose binding region or domain (CBD).

A cellulase composition produced by a naturally occurring source andwhich comprises one or more cellobiohydrolase type and endoglucanasetype components wherein each of these components is found at the ratioproduced by the source is sometimes referred to herein as a “completecellulase system” or a “complete cellulase composition” to distinguishit from the classifications and components of cellulase isolatedtherefrom, from incomplete cellulase compositions produced by bacteriaand some fungi, from a cellulase composition obtained from amicroorganism genetically modified so as to overproduce, underproduce,or not produce one or more of the cellobiohydrolase type and/orendoglucanase type components of cellulase, or from a truncatedcellulase enzyme composition, as defined herein.

The present invention specifically contemplates applicability tocellulases which contain core and binding domain regions as definedherein. For example, bacterial cellulases from Thermonospora sp.,Cellulomonas sp., Bacillus sp., Pseudomonas sp., Streptomyces sp. areknown to possess both a binding domain region and a core region.

Preferred for use in this invention are fungal cellulases. Morepreferably, the fungal cellulases are derived from Trichoderma sp.,including Trichoderma longibrachiatum, Trichoderma viride, Trichodermakoningii, Penicillium sp., Humicola, sp., including Humicola insolens,Aspergillus sp., and Fumarium sp. As used herein, the term “Trichoderma”or “Trichoderma sp.” refers to any fungal strains which have previouslybeen classified as Trichoderma or which are currently classified asTrichoderma. Most preferably, the cellulase's derived from Trichodermalongibrachiatum or Trichoderma viride.

“Fungal cellulase” means an enzyme composition derived from fungalsources or microorganisms genetically modified so as to incorporate andexpress all or part of the cellulase genes obtained from a fungalsource. Fungi capable of producing cellulases useful in preparingcellulase compositions described herein are disclosed in British PatentNo. 2 094 826A, the disclosure of which is incorporated herein byreference.

Most fungal cellulases generally have their optimum activity in theacidic or neutral pH range although some fungal cellulases are known topossess significant activity under neutral and slightly alkalineconditions, i.e., for example, cellulase derived from Humicola insolensis known to have activity in neutral to slightly alkaline conditions.

Fungal cellulases are known to be comprised of several enzymeclassifications having different substrate specificity, enzymatic actionpatterns, and the like. Additionally, enzyme components within eachclassification can exhibit different molecular weights, differentdegrees of glycosylation, different isoelectric points, differentsubstrate specificity etc. For example, fungal cellulases can containcellulase classifications which include endoglucanase type components(hereinafter “EG-type”), exo-cellobiohydrolase type components(hereinafter “CBH-type”), β-glucosidase type components (hereinafter“BG-type”), etc. On the other hand, while bacterial cellulases arereported in the literature as containing little or no CBH-typecomponents, there are a few cases where CBH-type components derived frombacterial cellulases have been reported to possess exo-cellobiohydrolaseactivity.

The fermentation procedures for culturing fungi and for production ofcellulase are known per se in the art. For example, cellulase systemscan be produced either by solid or submerged culture, including batch,fed-batch and continuous-flow processes. The collection and purificationof the cellulase systems from the fermentation broth can also beeffected by procedures known per se in the art.

“Endoglucanase-type components” or “EG-type” means fungal cellulasecomponents or a combination of components which exhibit textile activityproperties similar to the endoglucanase components of Trichodermalongibrachiatum (previously classified as Trichoderma reesei). In thisregard, the endoglucanase components of Trichoderma longibrachiatum (forexample, EGI, EGII, EGIII, and EGV, either alone or in combination) areknown to impart improved feel, improved appearance, softening, colorenhancement, and/or a stonewashed appearance to denim fabrics (ascompared to the fabric prior to treatment) when these components areincorporated into a textile treatment medium and the fabric is treatedwith this medium.

Accordingly, endoglucanase type components are those cellulasecomponents which impart specific enhancements to cellulose containingfabrics, such as improved feel, improved appearance, softening, colorenhancement, and/or a stonewashed appearance (as compared to the fabricbefore treatment) when these components are incorporated into a mediumused to treat the fabrics. Certain EG type components, including EGI,EGII and EGIII, may impart reduced strength loss to denim fabrics ascompared to the strength loss arising from treatment with a similarcellulase composition but which additionally contains CBH I typecomponents.

Endoglucanase type components as defined herein may not includecomponents traditionally classified as endoglucanases using activitytests such as the ability of the component (a) to hydrolyze solublecellulose derivatives such as carboxymethylcellulose (CMC), therebyreducing the viscosity of CMC containing solutions, (b) to readilyhydrolyze hydrated forms of cellulose such as phosphoric acid swollencellulose (e.g., Walseth cellulose) and hydrolyze less readily the morehighly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.).On the other hand, it is believed that not all endoglucanase components,as defined by such activity tests, will impart one or more of theenhancements to cellulose containing fabrics, including reduced strengthloss. Accordingly, for the purposes herein, it is appropriate to defineendoglucanase type components as those components of fungal cellulasewhich possess similar textile modification properties as possessed bythe endoglucanase components of Trichoderma longibrachiatum.

The different components generally have different properties, such asisoelectric point, molecular weight, degree of glycosylation, substratespecificity and enzymatic action patterns. The different isoelectricpoints of the components allow for their separation via techniques suchas ion exchange chromatography. In fact, the isolation of componentsfrom different sources is known in the art. See, for example, Bjork etal., U.S. Pat. No. 5,120,463; Schulein et al., International ApplicationWO 89/09259; Wood et al., Biochemistry and Genetics of CelluloseDegradation, pp. 31-52 (1988); Wood et al., Carbohydrate Research, Vol.190, pp. 279-297 (1989); Schulein, Methods in Enzymology, Vol. 160, pp.234-242 (1988); and the like. The entire disclosure of each of thesereferences is incorporated herein by reference.

The term “EGI” refers to an endoglucanase type component typicallyderived from, or embodying the identifying characteristics of thosederived from, EGI of Trichoderma sp. Thus, EGI refers to anendoglucanase derived from Trichoderma sp. characterized by a pH optimumof about 4.0 to 6.0, an isoelectric point (pI) of from about 4.5 to 4.7,and a molecular weight of about 47 to 49 Kdaltons. Preferably, EGI isderived from either Trichoderma longibrachiatum or from Trichodermaviride. EGI derived from Trichoderma longibrachiatum has a pH optimum ofabout 5.0, an isoelectric point (pI) of about 4.7 and a molecular weightof about 47 to 49 Kdaltons. EGI cellulase derived from Trichodermaviride has a pH optimum of about 5.0, an isoelectric point (pI) of about5.3 and a molecular weight of about 50 Kdaltons.

The term “EGII” as defined herein refers to an endoglucanase typecomponent typically derived from, or embodying the identifyingcharacteristics of those derived from, EGII of Trichoderma sp. It isnoted that EGII has been previously referred to by the nomenclature“EGIII” by some authors but current nomenclature uses the term EGII. Inany event, the EGII protein defined herein is substantially differentfrom the EGIII protein in its molecular weight, pI and pH optimum. Theterm “EGII cellulase” refers to the endoglucanase component derived fromTrichoderma spp. characterized by a pH optimum of about 4.0 to 6.0 anisoelectric point (pI) of from about 5.5, and a molecular weight ofabout 48 Kdaltons. Preferably, EGII cellulase is derived from eitherTrichoderma longibrachiatum or from Trichoderma viride.

The term “EGIII cellulase” as defined herein refers to an endoglucanasetype component typically derived from, or embodying the identifyingcharacteristics of those derived from, EGIII of Trichoderma sp. Thus,EGIII refers to the endoglucanase component derived from Trichodermaspp. characterized by a pH optimum of about 5.0 to 7.0, an isoelectricpoint (pI) of from about 7.2 to 8.0, and a molecular weight of about 23to 28 Kdaltons. Preferably, EGIII cellulase is derived from eitherTrichoderma longibrachiatum or from Trichoderma viride. EGIII cellulasederived from Trichoderma longibrachiatum has a pH optimum of about 5.5to 6.0, an isoelectric point (pI) of about 7.4 and a molecular weight ofabout 25 to 28 Kdaltons. EGIII cellulase derived from Trichoderma viridehas a pH optimum of about 5.5, an isoelectric point (pI) of about 7.7and a molecular weight of about 23.5 Kdaltons.

The term “EGV cellulase” as defined herein refers to an endoglucanasetype component typically derived from, or embodying the identifyingcharacteristics of those derived from, EGV of Trichoderma sp. Thus EGVrefers to the endoglucanase component derived from Trichoderma sp.characterized by Saloheimo et al., Molecular Microbiology 13 (2):219-228(1994); and Proceedings of the Second TRICEL Symposium on Trichodermareesei Cellulases and other Hydrolases, Esppo Finland 1993, ed. P.Suominen & J. T. Reinikainen. Foundation for Biotechnical and IndustrialFermentation Research 8, pp. 139-146 (1993).

“Exo-cellobiohydrolase type components” or “CBH-type” means fungalcellulase components which exhibit textile activity properties similarto CBH I and/or CBH II cellulase components of Trichodermalongibrachiatum. In this regard, when used in the absence of EG typecellulase components (as defined above), CBH I and CBH II components ofTrichoderma longibrachiatum alone are recognized as not imparting anysignificant enhancements in feel, appearance, softening colorenhancement and/or stonewashed appearance to denim fabrics.Additionally, when used in combination with some EG type components, ina ratio of approximately 2.5:1 of CBH I to EG components, the CBH Icomponent of Trichoderma longibrachiatum imparts enhanced strength lossto the denim fabrics.

Accordingly, CBH I type components and CBH II type components refer tothose fungal cellulase components which exhibit textile activityproperties similar to CBH I and CBH II components of Trichodermalongibrachiatum, respectively. As noted above, for CBH I typecomponents, this includes the property of increasing strength loss indenim fabrics treated with CBH I in the presence of EG type components.

Exo-cellobiohydrolase type components defined herein may not includecomponents traditionally classified as exo-cellobiohydrolases usingactivity tests such as those used to characterize CBH I and CBH II fromTrichoderma longibrachiatum. For example, exo-cellobiohydrolase typecomponents (a) are competitively inhibited by cellobiose (K_(i)approximately 1 mM); (b) are unable to hydrolyze substituted cellulosesto any significant degree, such as carboxymethylcellulose, and (c)hydrolyze phosphoric acid swollen cellulose and to a lesser degreehighly crystalline cellulose. On the other hand, it is believed thatsome cellulase components which are characterized as CBH type componentsby these activity tests, will impart improved feel, appearance,softening, color enhancement, and/or a stonewashed appearance tocellulose-containing fabrics when used alone in the cellulasecomposition. Accordingly, it is believed to be more accurate for thepurposes herein to define such exo-cellobiohydrolases as EG-typecomponents because these components possess similar functionalproperties in textile uses to those possessed by the endoglucanasecomponents of Trichoderma longibrachiatum.

In general, cellulase compositions comprising the various components ofa complete cellulase composition (“whole cellulose” can be obtained bypurification techniques based on their known characteristics andproperties. Specifically, the whole cellulase can be purified intosubstantially pure components by recognized separation techniquespublished in the literature, including ion exchange chromatography at asuitable pH, affinity chromatography, size exclusion and the like. Forexample, in ion exchange chromatography (usually anion exchangechromatography), it is possible to separate the cellulase components byeluting with a pH gradient, or a salt gradient, or both a pH and a saltgradient. After purification, the requisite amount of the desiredcomponents could be recombined.

The term “truncated cellulase”, as used herein, refers to a proteincomprising a truncated cellulase catalytic core or truncated cellulosebinding domain of exo-cellobiohydrolase or endoglucanase, for example,EGI type, EGII type, EGV type, CBHI type, CBHII type, or derivativesthereof. As stated above, many cellulase enzymes are believed to bebifunctional in that they contain domains which are directed toward bothcatalytic or hydrolytic activity with respect to the cellulosesubstrate, and also non-catalytic cellulose binding activity. Thus, atruncated cellulase is a cellulase which in an intact form, containsboth a core and a binding domain but which is treated so as to lack oneor the other domain.

The catalytic core and the cellulose binding domain of a cellulaseenzyme are believed to act together in a synergistic manner to effectefficient and often deleterious hydrolysis of cellulose fibers in acellulose containing fabric. Further, cellulase catalytic activity andcellulose binding activity are believed to be specific to distinctstructural domains. For example, as indicated above, many cellulaseenzymes, including cellulases from, for example, T. longibrachiatum andHumicola insolens are known to incorporate a catalytic core domainsubunit which is attached via a linker region to a cellulose bindingdomain subunit.

A “truncated cellulase derivative” encompasses a truncated cellulasecore or truncated cellulose binding domain, as defined herein, whereinthere may be an addition or deletion of one or more amino acids toeither or both of the C- and N-terminal ends of the truncated cellulase,or a substitution, insertion or deletion of one or more amino acids atone or more sites throughout the truncated cellulase. Derivativesinclude mutants that preserve their character as truncated cellulasecore or truncated cellulose binding domain, as defined below. It is alsointended by the term “derivative of a truncated cellulase” to includecore or binding domains of the exoglucanase or endoglucanase enzymesthat have attached thereto one or more amino acids from the linkerregion.

A truncated cellulase derivative further refers to a proteinsubstantially similar in structure and biological activity to atruncated cellulase core or truncated cellulose binding domain protein,but which has been genetically engineered to contain a modified aminoacid sequence. Thus, provided that the two proteins possesssubstantially similar activity, they are considered “derivatives” evenif the primary structure of one protein does not possess the identicalamino acid sequence to that found in the other.

It is contemplated that a truncated cellulase derivative may be derivedfrom a DNA fragment encoding a catalytic truncated core or a truncatedcellulose binding domain which further contains an addition of one ormore nucleotides internally or at the 5′ or 3′ end of the DNA fragment,a deletion of one or more nucleotides internally or at the 5′ or 3′ endof the DNA fragment or a substitution of one or more nucleotidesinternally or at the 5′ or 3′ end of the DNA fragment wherein thefunctional activity of the expressed truncated cellulose binding orcatalytic core domain (truncated cellulase derivative) is retained. ADNA fragment encoding a cellulase catalytic core or cellulose bindingdomain may further include a linker or hinge DNA sequence or portionthereof attached to the core or binding domain DNA sequence at eitherthe 5′ or 3′ end wherein the functional activity of the encodedtruncated cellulose binding domain or cellulase core domain (truncatedcellulase derivative) is retained.

The term “truncated cellulase core” or “truncated cellulase region”refers herein to a peptide comprising the catalytic core domain ofexo-cellobiohydrolase or endoglucanase, for example, EGI type, EGIItype, EGV type, CBHI type or CBHII type, or a derivative thereof that iscapable of enzymatically cleaving cellulose polymers, including but notlimited to pulp or phosphoric acid swollen cellulose. However, atruncated cellulase core will not possess cellulose binding activityattributable to a cellulose binding domain. A truncated cellulase coreis distinguished from a non-truncated cellulase which, in an intactform, possesses poor cellulose binding activity. A truncated cellulasecore may include other entities which do not include cellulose bindingactivity attributable to a cellulose binding domain. For example, thepresence of a linker or hinge is specifically contemplated. Similarly,the covalent attachment of another enzymatic entity to the truncatedcellulase core is also specifically contemplated.

The performance (or activity) of a protein containing a truncatedcatalytic core or a derivative thereof may be determined by methods wellknown in the art. (See Wood, T. M. et al in Methods in Enzymology, Vol.160, Editors: Wood, W. A. and Kellogg, S. T., Academic Press, pp.87-116, 1988) For example, such activities can be determined byhydrolysis of phosphoric acid-swollen cellulose and/or solubleoligosaccharides followed by quantification of the reducing sugarsreleased. In this case the soluble sugar products, released by theaction of CBH or EG cellulase core domains or derivatives thereof, canbe detected by HPLC analysis or by use of colorimetric assays formeasuring reducing sugars. It is expected that these catalytic domainsor derivatives thereof will retain at least 10% of the activityexhibited by the intact enzyme when each is assayed under similarconditions and dosed based on similar amounts of catalytic domainprotein.

The term “truncated cellulose binding domain” refers herein to a peptideor group of related peptides comprising cellulose binding activity of anexo-cellobiohydrolase or an endoglucanase, for example, EGI type, EGIItype, EGV type, CBHI type or CBHII type, or a derivative thereof thatnon-covalently binds to a polysaccharide such as cellulose. It isbelieved that cellulose binding domains attach the enzyme to celluloseand function independently from the catalytic core of the cellulaseenzyme. A truncated cellulose binding domain will not possess thesignificant hydrolytic activity attributable to a catalytic core. Atruncated cellulose binding domain is distinguished from a non-truncatedcellulase which, in an intact form, possesses no cellulase catalyticcore. A truncated cellulose binding domain may include other entitieswhich do not include cellulose cleavage activity attributable tocellulase catalytic core. For example, the presence of a linker or hingeis specifically contemplated. Similarly, the covalent attachment ofanother enzymatic entity to the truncated cellulose binding domain isalso specifically contemplated.

The performance (or activity) of a truncated cellulose binding domain orderivatives thereof as described in the present invention may bedetermined by cellulose binding assays using cellulose substrates suchas avicel, pulp or cotton. It is expected that these novel truncatedcellulose binding domains or derivatives thereof will retain at least10% of the binding affinity compared to that exhibited by thenon-truncated enzyme when each is assayed under similar conditions anddosed based on similar amounts of binding domain protein. The amount ofnon-bound binding domain may be quantified by direct protein analysis,by chromatographic methods, or possibly by immunological methods.

Other methods well known in the art that measure cellulase catalyticand/or binding activity via the physical or chemical properties ofparticular treated substrates may also be suitable in the presentinvention. For example, for methods that measure physical properties ofa treated substrate, the substrate is analyzed for modification ofshape, texture, surface, or structural properties, modification of the“wet” ability, e.g. substrates ability to absorb water, or modificationof swelling. Other parameters which may determine activity include themeasuring of the change in the chemical properties of treated solidsubstrates. For example, the diffusion properties of dyes or chemicalsmay be examined after treatment of solid substrate with the truncatedcellulase binding protein or derivatives thereof described in thepresent invention. Appropriate substrates for evaluating activityinclude Avicel, rayon, pulp fibers, cotton or ramie fibers, paper, kraftor ground wood pulp, for example. (See also Wood, T. M. et al in“Methods in Enzymology”, Vol. 160, Editors: Wood, W. A. and Kellogg, S.T., Academic Press, pp. 87-116, (1988)).

In addition to truncated cellulase core, naturally occurring cellulaseswhich lack a binding domain possess the advantages of the truncatedcatalytic cores defined herein. For example, bacterial cellulase derivedfrom Erwinia carotovora are believed to possess no binding domain, (seee.g., Saarilahti, et al. Gene, Vol. 90, pp. 9-14 (1990)). Similarly,cellulases derived from Clostridium thermocellum, also are believed topossess no binding domain (see e.g. Gilkes, et al., MicrobiologicalReviews, pp. 303-315 (1991)). Thus, the use of naturally occurringcellulases having no binding domain will provide decreased backstainingat equivalent levels of abrasion to known cellulases.

“β-Glucosidase (BG) components” means components of a complete cellulasecomposition which exhibit BG activity; that is to say that suchcomponents will act from the non-reducing end of cellobiose and othersoluble cellooligosaccharides (“cellobiose”) and give glucose as thesole product. BG components do not adsorb onto or react with cellulosepolymers. Furthermore, such BG components are competitively inhibited byglucose (K_(i) approximately 1 mM). While in a strict sense, BGcomponents are not literally cellulases because they cannot degradecellulose, such BG components are included within the definition of thecellulase system because these enzymes facilitate the overalldegradation of cellulose by further degrading the inhibitory cellulosedegradation products (particularly cellobiose) produced by the combinedaction of CBH type components and EG type components.

Methods to either increase or decrease the amount of BG components inthe cellulase composition is disclosed in WO 92/10581 which applicationis incorporated herein by reference in its entirety.

“Linker or hinge region” means a short peptide region that linkstogether structurally distinct catalytic core and cellulose bindingdomains of a cellulase. These domains in T. longibrachiatum cellulases,for example, are linked by a peptide rich in Ser, Thr and Pro.

“Signal sequence” means a sequence of amino acids bound to theN-terminal portion of a protein which facilitates the secretion of themature form of the protein outside of the cell. This definition of asignal sequence is a functional one. The mature form of theextracellular protein lacks the signal sequence which is cleaved offduring the secretion process.

“Host cell” means a cell which acts as a host for a recombinant DNAvector according to the present invention. In a preferred embodimentaccording to the present invention, “host cell” means both the cells andprotoplasts created from the cells of Trichoderma sp.

“DNA construct or vector” (used interchangeably herein) means anucleotide sequence which comprises one or more DNA fragments or DNAvariant fragments encoding any of the novel truncated cellulases orderivatives described above.

“Functionally attached to” means that a regulatory region, such as apromoter, terminator, secretion signal or enhancer region is attached toa structural gene and controls the expression of that gene.

“Buffer” means art recognized acid/base reagents which stabilize thecellulase solution against undesired pH shifts during cellulasetreatment of the cellulose containing fabric. In this regard, it is artrecognized that cellulase activity is pH dependent. For example, aspecific cellulase composition will exhibit cellulolytic activity withina defined pH range with optimal cellulolytic activity generally beingfound within a small portion of this defined range. The specific pHrange for cellulolytic activity will vary with each cellulasecomposition. As noted above, while most cellulases will exhibitcellulolytic activity within an acidic to neutral pH profile, there aresome cellulase compositions which exhibit cellulolytic activity in analkaline pH environment.

During cellulase treatment of the cellulose containing fabric, it ispossible that the pH of the initial cellulase solution could be outsidethe range required for cellulase activity. It is further possible forthe pH to change during treatment of the cellulose containing fabric,for example, by the generation of a reaction product which alters the pHof the solution. In either event, the pH of an unbuffered cellulasesolution could be outside the range required for cellulolytic activity.When this occurs, undesired reduction or cessation of cellulolyticactivity in the cellulase solution occurs.

In view of the above, the pH of the cellulase solution should bemaintained within the range required for cellulolytic activity. Onemeans of accomplishing this is by simply monitoring the pH of the systemand adjusting the pH as required by the addition of either an acid or abase. However, in a preferred embodiment, the pH of the system ispreferably maintained within the desired pH range by the use of a bufferin the cellulase solution. In general, a sufficient amount of buffer isemployed so as to maintain the pH of the solution within the rangewherein the employed cellulase exhibits activity. Insofar as differentcellulase compositions have different pH ranges for exhibiting cellulaseactivity, the specific buffer employed is selected in relationship tothe specific cellulase composition employed. The buffer(s) selected foruse with the cellulase composition employed can be readily determined bythe skilled artisan taking into account the pH range and optimum for thecellulase composition employed as well as the pH of the cellulasesolution. Preferably, the buffer employed is one which is compatiblewith the cellulase composition and which will maintain the pH of thecellulase solution within the pH range required for optimal activity.Suitable buffers include sodium citrate, ammonium acetate, sodiumacetate, disodium phosphate, and any other art recognized buffers.

II. Preparation of Truncated Cellulase Enzymes

The present invention relates to the use of truncated cellulases andderivatives of truncated cellulases. These enzymes are preferablyprepared by recombinant methods. Additionally, truncated cellulaseproteins for use in the present invention may be obtained by other artrecognized means such as chemical cleavage or proteolysis of completecellulase protein.

A preferred mode for preparing truncated cellulase according to thepresent invention comprises obtaining a modified Trichoderma sp. hostcell that is missing one or more cellulase activities. The modifiedTrichoderma sp. cell is transformed with a DNA construct comprising atleast a fragment of DNA encoding a portion or all of the binding or coreregion of a exo-cellobiohydrolase or an endoglucanase, for example, EGI,EGII, EGV, CBHI or CBHII functionally attached to a promoter. Thetransformed host cell is then grown under conditions so as to expressthe desired protein. Subsequently, the desired protein product ispurified to substantial homogeneity.

Preferably, the microorganism to be transformed comprises a strainderived from Trichoderma sp. More preferably, the strain comprises T.longibrachiatum cellulase over-producing strain. For example, RL-P37,described by Sheir-Neiss et al. in Appl. Microbiol. Biotechnology, 20(1984) pp. 46-53 is known to secrete elevated amounts of cellulaseenzymes. Functional equivalents of RL-P37 include Trichodermalongibrachiatum strain RUT-C30 (deposited under deposit no. ATCC No.56765) and ATCC deposit nos. 58351, 58352, and 58353.

Still more preferably, the Trichoderma host cell strains have had one ormore cellulase genes deleted prior to introduction of a DNA construct orplasmid containing the DNA fragment encoding the truncated cellulaseprotein. Such strains may be prepared by the method disclosed in U.S.Pat. No. 5,246,853 and WO 92/06209, which disclosures are herebyincorporated by reference. By expressing a truncated cellulase in a hostmicroorganism that is missing one or more cellulase genes, theidentification and subsequent purification procedures are simplified.Any gene from Trichoderma sp. which has been cloned can be deleted, forexample, the cbh1, cbh2, egl1, and egl3 genes.

Gene deletion may be accomplished by inserting a form of the desiredgene to be deleted or disrupted into a plasmid by methods known in theart. An appropriate deletion plasmid will generally have uniquerestriction enzyme sites present therein to enable the fragment ofhomologous Trichoderma sp. DNA to be removed as a single linear piece.The deletion plasmid is then cut at an appropriate restriction enzymesite(s), internal to the coding region, and the gene coding sequence orpart thereof replaced with a selectable marker. Flanking DNA sequencesfrom the locus of the gene to be deleted or disrupted, preferablybetween about 0.5 to 2.0 kb, remain on either side of the selectablemarker gene.

A selectable marker must be chosen so as to enable detection of thetransformed fungus. Any selectable marker gene which is expressed in theselected microorganism will be suitable. For example, with Trichodermasp., the selectable marker is chosen so that the presence of theselectable marker in the transformants will not significantly affect theproperties thereof. Such a selectable marker may be a gene which encodesan assayable product. For example, a functional copy of a Trichoderma spgene may be used which if lacking in the host strain results in the hoststrain displaying an auxotrophic phenotype.

In a preferred embodiment, a pyr4⁻ derivative strain of Trichoderma sp.is transformed so that one or more cellulase genes are replaced by thepyr4 gene, thus providing a selectable marker. A pyr4⁻ derivative strainmay be obtained by subjecting Trichoderma sp. strains to fluorooroticacid (FOA). The pyr4 gene encodes orotidine-5′-monophosphatedecarboxylase, an enzyme required for the biosynthesis of uridine.Strains with an intact pyr4 gene grow in a medium lacking uridine butare sensitive to fluoroorotic acid. It is possible to select pyr4⁻derivative strains which lack a functional orotidine monophosphatedecarboxylase enzyme and require uridine for growth by selecting for FOAresistance. Using the FOA selection technique it is also possible toobtain uridine requiring strains which lack a functional orotatepyrophosphoribosyl transferase. It is possible to transform these cellswith a functional copy of the gene encoding this enzyme (Berges andBarreau, 1991, Curr. Genet. 19 pp359-365). Selection of derivativestrains is easily performed using the FOA resistance technique referredto above, and thus, the pyr4 gene is preferably employed as a selectablemarker.

To transform pyr4⁻ Trichoderma sp. so as to be lacking in the ability toexpress one or more cellulase genes, a single DNA fragment comprising adisrupted or deleted cellulase gene is then isolated from the deletionplasmid and used to transform an appropriate pyr⁻ Trichoderma host.Transformants are then identified and selected based on their ability toexpress the pyr4 gene product and thus compliment the uridine auxotrophyof the host strain. Southern blot analysis is then carried out on theresultant transformants to identify and confirm a double cross overintegration event which replaces part or all of the coding region of thegene to be deleted with the pyr4 selectable markers.

Although the specific plasmid vectors described above relate topreparation of pyr⁻ transformants, the present invention is not limitedto these vectors. Various genes can be deleted and replaced in theTrichoderma sp. strain using the above techniques. In addition, anyavailable selectable markers can be used, as discussed above. In fact,any Trichoderma sp. gene which has been cloned, and thus identified, canbe deleted from the genome using the above-described strategy.

As stated above, the host strains used are derivatives of Trichodermasp. which lack or have a nonfunctional gene or genes corresponding tothe selectable marker chosen. For example, if the selectable marker ofpyr4 is chosen, then a specific pyr derivative strain is used as arecipient in the transformation procedure. Similarly, selectable markerscomprising Trichoderma sp. genes equivalent to the Aspergillus nidulansgenes argB, trpC, niaD may be used. The corresponding recipient strainmust therefore be a derivative strain such as argB⁻, trpC⁻, niaD⁻,respectively.

DNA encoding the truncated cellulase protein is then prepared forinsertion into an appropriate microorganism. According to the presentinvention, DNA encoding for a truncated cellulase enzyme comprises DNAencoding for a protein which corresponds to the catalytic core region ofthe cellulase enzyme. Accordingly, DNA may be derived from any microbialsource which is known to produce cellulase, where the gene is identifiedand isolated. In a preferred embodiment, the DNA encodes for a truncatedcellulase protein derived from Trichoderma sp., and more preferably fromTrichoderma longibrachiatum. Thus, the DNA may encode for an EGI, EGII,CBHI or CBHII core protein. Preferably, the DNA gene fragment or variantDNA fragment codes for the core or binding domains of a Trichoderma sp.cellulase or derivative thereof that additionally retains the functionalactivity of the truncated core or binding domain, respectively. Further,the DNA fragment or variant thereof comprising the sequence of the coreor binding domain regions may additionally have attached thereto alinker, or hinge region DNA sequence or portion thereof wherein theencoded truncated cellulase still retains either cellulase core orbinding domain activity, respectively.

The DNA fragment or DNA variant fragment encoding the truncatedcellulase or derivative may be functionally attached to a fungalpromoter sequence, for example, the promoter of the cbh1 or egl1 gene.It is also contemplated that more than one copy of DNA encoding atruncated cellulase may be recombined into the strain.

The DNA encoding the truncated cellulase protein may be prepared by theconstruction of an expression vector carrying the DNA encoding thetruncated cellulase. The expression vector carrying the inserted DNAfragment encoding the truncated cellulase may be any vector which iscapable of replicating autonomously in a given host organism, typicallya plasmid. In preferred embodiments two types of expression vectors forobtaining expression of genes or truncations thereof are contemplated.The first contains DNA sequences in which the promoter, gene codingregion, and terminator sequence all originate from the gene to beexpressed. The gene truncation is obtained by deleting away theundesired DNA sequences (coding for unwanted domains) to leave thedomain to be expressed under control of its own transcriptional andtranslational regulatory sequences. A selectable marker is alsocontained on the vector allowing the selection for integration into thehost of multiple copies of the novel gene sequences.

For example, pEGIΔ3′ pyr contains the EGI cellulase core domain underthe control of the EGI promoter, terminator, and signal sequences. The3′ end on the EGI coding region containing the cellulose binding domainhas been deleted. The plasmid also contains the pyr4 gene for thepurpose of selection.

The second type of expression vector is preassembled and containssequences required for high level transcription and a selectable marker.It is contemplated that the coding region for a gene or part thereof canbe inserted into this general purpose expression vector such that it isunder the transcriptional control of the expression cassettes promoterand terminator sequences.

For example, PTEX is such a general purpose expression vector. Genes orpart thereof can be inserted downstream of the strong CBHI promoter. TheExamples disclosed herein are included in which cellulase catalytic coreand binding domains are shown to be expressed using this system.

In the vector, the DNA sequence encoding the truncated cellulase orother novel proteins of the present invention should be operably linkedto transcriptional and translational sequences, i.e., a suitablepromoter sequence and signal sequence in reading frame to the structuralgene. The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell and may be derived from genes encodingproteins either homologous or heterologous to the host cell. The signalpeptide provides for extracellular expression of the truncated cellulaseor derivatives thereof. The DNA signal sequence is preferably the signalsequence naturally associated with the truncated gene to be expressed,however the signal sequence from any exo-cellobiohydrolases orendoglucanase is contemplated in the present invention.

The procedures used to ligate the DNA sequences coding for the truncatedcellulases, derivatives thereof or other novel cellulases of the presentinvention with the promoter, and insertion into suitable vectorscontaining the necessary information for replication in the host cellare well known in the art.

The DNA vector or construct described above may be introduced in thehost cell in accordance with known techniques such as transformation,transfection, microinjection, microporation, biolistic bombardment andthe like.

In the preferred transformation technique, it must be taken into accountthat the permeability of the cell wall in Trichoderma sp. is very low.Accordingly, uptake of the desired DNA sequence, gene or gene fragmentis at best minimal. There are a number of methods to increase thepermeability of the Trichoderma sp. cell wall in the derivative strain(i.e., lacking a functional gene corresponding to the used selectablemarker) prior to the transformation process.

The preferred method in the present invention to prepare Trichoderma sp.for transformation involves the preparation of protoplasts from fungalmycelium. The mycelium can be obtained from germinated vegetativespores. The mycelium is treated with an enzyme which digests the cellwall resulting in protoplasts. The protoplasts are then protected by thepresence of an osmotic stabilizer in the suspending medium. Thesestabilizers include sorbitol, mannitol, potassium chloride, magnesiumsulfate and the like. Usually the concentration of these stabilizersvaries between 0.8 M to 1.2 M. It is preferable to use about a 1.2 Msolution of sorbitol in the suspension medium.

Uptake of the DNA into the host Trichoderma sp. strain is dependent uponthe calcium ion concentration. Generally between about 10 Mm CaCl₂ and50 Mm CaCl₂ is used in an uptake solution. Besides the need for thecalcium ion in the uptake solution, other items generally included are abuffering system such as TE buffer (10 Mm Tris, Ph 7.4; 1 Mm EDTA) or 10Mm MOPS, Ph 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). It is believed that the polyethylene glycol acts to fusethe cell membranes thus permitting the contents of the medium to bedelivered into the cytoplasm of the Trichoderma sp. strain and theplasmid DNA is transferred to the nucleus. This fusion frequently leavesmultiple copies of the plasmid DNA tandemly integrated into the hostchromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁸to 10⁹/ml, preferably 2×10⁸/ml are used in transformation. Theseprotoplasts or cells are added to the uptake solution, along with thedesired linearized selectable marker having substantially homologousflanking regions on either side of said marker to form a transformationmixture. Generally a high concentration of PEG is added to the uptakesolution. From 0.1 to 1 volume of 25% PEG 4000 can be added to theprotoplast suspension. However, it is preferable to add about 0.25volumes to the protoplast suspension. Additives such as dimethylsulfoxide, heparin, spermidine, potassium chloride and the like may alsobe added to the uptake solution and aid in transformation.

Generally, the mixture is then incubated at approximately 0° C. for aperiod between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired gene or DNAsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is preferably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then incubated at room temperature beforethe addition of a sorbitol and CaCl₂ solution. The protoplast suspensionis then further added to molten aliquots of a growth medium. This growthmedium permits the growth of transformants only. Any growth medium canbe used in the present invention that is suitable to grow the desiredtransformants. However, if Pyr⁺ transformants are being selected it ispreferable to use a growth medium that contains no uridine. Thesubsequent colonies are transferred and purified on a growth mediumdepleted of uridine.

At this stage, stable transformants were distinguished from unstabletransformants by their faster growth rate and the formation of circularcolonies with a smooth, rather than ragged outline on solid culturemedium lacking uridine. Additionally, in some cases a further test ofstability was made by growing the transformants on solid non-selectivemedium (i.e. containing uridine), harvesting spores from this culturemedium and determining the percentage of these spores which willsubsequently germinate and grow on selective medium lacking uridine.

In a particular embodiment of the above method, the truncated cellulasesor derivatives thereof are recovered in active form from the host celleither as a result of the appropriate post translational processing ofthe novel truncated cellulase or derivative thereof.

The truncated cellulases are recovered from the medium by conventionaltechniques including separations of the cells from the medium bycentrifugation, filtration, and precipitation of the proteins in thesupernatant or filtrate with a salt, for example, ammonium sulphate.Additionally, chromatography procedures such as ion exchangechromatography, affinity chromatography may be used. Alternatively, thesecreted protein product may be isolated and purified by binding to apolysaccharide substrate or antibody matrix. The antibodies (polyclonalor monoclonal) may be raised against cellulase core or binding domainpeptides, or synthetic peptides may be prepared from portions of thecore domain or binding domain and used to raise polyclonal antibodies.

The DNA transformed into the host cell may comprise additionalembodiments. For example, cellulase enzymes are contemplated whichcombine a core region derived from either a homologous or heterologousmicrobial source.

III. Methods Of Treating Cellulose Containing Fabric Using TruncatedCellulase Enzymes

As noted above, the present invention pertains to methods for treatingcellulose containing fabrics with a truncated cellulase enzyme. The useof the truncated cellulase composition of this invention provides thenovel and surprising result of effecting a relatively low level of dyeredeposition while maintaining an equivalent level of abrasion comparedto prior art cellulase treatment. Because the level of abrasion acts asan indicator of the quality and effectiveness of particular cellulasetreatment techniques, e.g., stonewashing or laundering, the use of theinstant invention provides a surprisingly high quality textile treatmentcomposition. In the laundering context, abrasion is sometimes referredto as color clarification, defuzzing or biopolishing.

The present invention specifically contemplates the use of truncatedcellulase core, alone or in combination with additional cellulasecomponents, to achieve excellent abrasion with reduced redeposition whencompared to non-truncated cellulase. Additionally, naturally occurringcellulase enzymes which lack a binding domain are contemplated as withinthe scope of the invention. It is also contemplated that the methods ofthis invention will provide additional enhancements to treated cellulosecontaining fabric, including improvements in the feel and/or appearanceof the fabric.

A. Methodology for Stonewashing with Truncated Cellulase Compositions

According to the present invention, the truncated cellulase compositionsdescribed above may be employed as a stonewashing composition.Preferably, the stonewashing composition of the instant comprises anaqueous solution which contains an effective amount of a truncatedcellulase together with other optional ingredients including, forexample, a buffer, a surfactant, and a scouring agent.

An effective amount of truncated cellulase enzyme composition is aconcentration of truncated cellulase enzyme sufficient for its intendedpurpose. Thus an “effective amount” of truncated cellulase in thestonewashing composition according to the present invention is thatamount which will provide the desired treatment, e.g., stonewashing. Theamount of truncated cellulase employed is also dependent on theequipment employed, the process parameters employed (the temperature ofthe truncated cellulase treatment solution, the exposure time to thecellulase solution, and the like), and the cellulase activity (e.g., aparticular solution will require a lower concentration of cellulasewhere a more active cellulase composition is used as compared to a lessactive cellulase composition). The exact concentration of truncatedcellulase can be readily determined by the skilled artisan based on theabove factors as well as the desired result. Preferably the truncatedcellulase composition is present in a concentration of from 1-1000 ppm,more preferably 10-400 ppm and most preferably 20-100 ppm total protein.

Optionally, a buffer is employed in the stonewashing composition suchthat the concentration of buffer is that which is sufficient to maintainthe pH of the solution within the range wherein the employed truncatedcellulase exhibits activity which, in turn, depends on the nature of thetruncated cellulase employed. The exact concentration of buffer employedwill depend on several factors which the skilled artisan can readilytake into account. For example, in a preferred embodiment, the buffer aswell as the buffer concentration are selected so as to maintain the pHof the final truncated cellulase solution within the pH range requiredfor optimal cellulase activity. Preferably, buffer concentration in thestonewashing composition is about 0.001N or greater. Suitable buffersinclude, for example; citrate and acetate.

In addition to truncated cellulase and a buffer, the stonewashingcomposition may optionally contain a surfactant. Preferably, thesurfactant is present in a concentration in the diluted wash mediums ofgreater than 100 ppm, preferably from about 200-15,000 ppm. Suitablesurfactants include any surfactant compatible with the cellulase and thefabric including, for example, anionic, non-ionic and ampholyticsurfactants. Suitable anionic surfactants for use herein include linearor branched alkylbenzenesulfonates; alkyl or alkenyl ether sulfateshaving linear or branched alkyl groups or alkenyl groups; alkyl oralkenyl sulfates; olefinsulfonates; alkanesulfonates and the like.Suitable counter ions for anionic surfactants include alkali metal ionssuch as sodium and potassium; alkaline earth metal ions such as calciumand magnesium; ammonium ion; and alkanolamines having 1 to 3 alkanolgroups of carbon number 2 or 3. Ampholytic surfactants includequaternary ammonium salt sulfonates, and betaine-type ampholyticsurfactants. Such ampholytic surfactants have both the positive andnegative charged groups in the same molecule. Nonionic surfactantsgenerally comprise polyoxyalkylene ethers, as well as higher fatty acidalkanolamides or alkylene oxide adduct thereof, and fatty acid glycerinemonoesters. Mixtures of surfactants can also be employed in mannersknown in the art.

In a preferred embodiment, a concentrated stonewashing composition canbe prepared for use in the methods described herein. Such concentrateswould contain concentrated amounts of the truncated cellulasecomposition described above, buffer and surfactant, preferably in anaqueous solution. When so formulated, the stonewashing concentrate canreadily be diluted with water so as to quickly and accurately preparestonewashing compositions according to the present invention and havingthe requisite concentration of these additives. Preferably, suchconcentrates will comprise from about 0.1 to about 50 weight percent ofa fungal cellulase composition described above (protein); from about 0.1to about 80 weight percent buffer; from about 0 to about 50 weightpercent surfactant; with the balance being water. When aqueousconcentrates are formulated, these concentrates can be diluted so as toarrive at the requisite concentration of the components in the truncatedcellulase solution as indicated above. As is readily apparent, suchstonewashing concentrates will permit facile formulation of thetruncated cellulase solutions as well as permit feasible transportationof the concentration to the location where it will be used. Thestonewashing concentrate can be in any art recognized form, for example,liquid, emulsion, gel, or paste. Such forms are well known to theskilled artisan.

When a solid stonewashing concentrate is employed, the cellulasecomposition may be a granule, a powder, an agglomerate or a solid disk.When granules are used, the granules are preferably formulated so as tocontain a cellulase protecting agent. See, for instance, U.S. Ser. No.07/642,669, filed Jan. 17, 1991 and entitled “GRANULES CONTAINING BOTHAN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONSCONTAINING SUCH GRANULES,” which application is incorporated herein byreference in its entirety. Likewise, the granules can be formulated soas to contain materials to reduce the rate of dissolution of thegranules into the wash medium. Such materials and granules are disclosedin U.S. Pat. No. 5,254,283 which is incorporated herein by reference inits entirety.

Other materials can also be used with or placed in the stonewashingcomposition of the present invention as desired, including stones,pumice, fillers, solvents, enzyme activators, and otheranti-redeposition agents.

The cellulose containing fabric is contacted with the stonewashingcomposition containing an effective amount of the truncated cellulaseenzyme or derivative by intermingling the treating composition with thestonewashing composition, and thus bringing the truncated cellulaseenzyme into proximity with the fabric. For example, if the treatingcomposition is an aqueous solution, the fabric may be directly soaked inthe solution. Similarly, where the stonewashing composition is aconcentrate, the concentrate is diluted into a water bath with thecellulose containing fabric. When the stonewashing composition is in asolid form, for example a pre-wash gel or solid stick, the stonewashingcomposition may be contacted by directly applying the composition to thefabric or to the wash liquor.

The cellulose containing fabric is incubated with the stonewashingsolution under conditions effective to allow the enzymatic action toconfer a stonewashed appearance to the cellulose containing fabric. Forexample, during stonewashing, the pH, liquor ratio, temperature andreaction time may be adjusted to optimize the conditions under which thestonewashing composition acts. “Effective conditions” necessarily refersto the pH, liquor ratio, and temperature which allow the truncatedcellulase enzyme to react efficiently with cellulose containing fabric.The reaction conditions for truncated cellulase core, and thus theconditions effective for the stonewashing compositions of the presentinvention, are substantially similar to well known methods used withcorresponding non-truncated cellulases. Accordingly, the conditionseffective for treatment of cellulose containing fabric with astonewashing composition comprising CBHI type core according to thepresent invention are substantially similar to those in the prior artusing wild-type CBHI type cellulase compositions. Similarly, where amixture of truncated and non-truncated cellulase is utilized, theconditions should be optimized similar to where a similar combinationmay have been used. Accordingly, it is within the skill of those in theart to maximize conditions for using the stonewashing compositionsaccording to the present invention.

The liquor ratios during stonewashing, i.e., the ratio of weight ofstonewashing composition solution (i.e., the wash liquor) to the weightof fabric, employed herein is generally an amount sufficient to achievethe desired stonewashing effect in the denim fabric and is dependentupon the process used. Preferably, the liquor ratios are from about 4:1to about 50:1; more preferably from 5:1 to about 20:1, and mostpreferably from about 10:1 to about 15:1.

Reaction temperatures during stonewashing with the present stonewashingcompositions are governed by two competing factors. Firstly, highertemperatures generally correspond to enhanced reaction kinetics, i.e.,faster reactions, which permit reduced reaction times as compared toreaction times required at lower temperatures. Accordingly, reactiontemperatures are generally at least about 10° C. and greater. Secondly,cellulase is a protein which loses activity beyond a given reactiontemperature which temperature is dependent on the nature of thecellulase used. Thus, if the reaction temperature is permitted to go toohigh, then the cellulolytic activity is lost as a result of thedenaturing of the cellulase. As a result, the maximum reactiontemperatures employed herein are generally about 65° C. In view of theabove, reaction temperatures are generally from about 30° C. to about65° C.; preferably, from about 35° C. to about 60° C.; and morepreferably, from about 35° C. to about 55° C.

Reaction times are dependent on the specific conditions under which thestonewashing occurs. For example, pH, temperature and concentration oftruncated cellulase will all effect the optimal reaction time.Generally, reaction times are from about 5 minutes to about 5 hours, andpreferably from about 10 minutes to about 3 hours and, more preferably,from about 20 minutes to about 1 hour.

Cellulose containing fabrics treated in the stonewashing methodsdescribed above using truncated cellulase compositions according to thepresent invention show reduced redeposition of dye as compared to thesame cellulose containing fabrics treated in the same manner with annon-truncated cellulase composition.

B. Methodology for Treating Cellulose Containing Fabrics with aDeterrent Composition Comprising Truncated Cellulase Enzyme

According to the present invention, the truncated cellulase compositionsdescribed above may be employed as detergent composition. The detergentcompositions according to the present invention are useful as pre-washcompositions, pre-soak compositions, or for detergent cleaning duringthe regular wash cycle. Preferably, the detergent composition of thepresent invention comprises an effective amount of truncated cellulase,and a surfactant, and optionally include other ingredients describedbelow.

An effective amount of truncated cellulase employed in the detergentcompositions of this invention is an amount sufficient to impartimproved abrasion to cellulase containing fabrics. Preferably, thetruncated cellulase employed is in a concentration of about 0.001% toabout 25%, more preferably, about 0.02% to about 10% by weight percentof detergent.

The specific concentration of truncated cellulase enzyme employed in thedetergent composition is preferably selected so that upon dilution intoa wash medium, the concentration of truncated cellulase enzyme is in arange of about 0.1 to about 1000 ppm, preferably from about 0.2 ppm toabout 500 ppm, and most preferably from about 0.5 ppm to about 250 ppmtotal protein. Thus, the specific amount of truncated cellulase enzymeemployed in the detergent composition will depend on the extent to whichthe detergent will be diluted upon addition to water so as to form awash solution.

At lower concentrations of truncated cellulase enzyme, i.e.,concentrations of truncated enzyme lower than 20 ppm, the decreasedbackstaining or redeposition with equivalent surface fiber abrasion whencompared to prior art compositions will become evident after repeatedwashings. At higher concentrations, i.e., concentrations of truncatedcellulase enzymes of greater than 40 ppm, the decreased backstainingwith equivalent surface fiber removal will become evident after a singlewash.

The detergent compositions of the present invention may be in any artrecognized form, for example, as a liquid diluent, in granules, inemulsions, in gels, or in pastes. Such forms are well known to theskilled artisan. When a solid detergent composition is employed, thetruncated cellulase is preferably formulated as granules. Preferably,the granules can be formulated so as to additionally contain a cellulaseprotecting agent. See, for instance, U.S. Ser. No. 07/642,669 filed Jan.17, 1991 and entitled “GRANULES CONTAINING BOTH AN ENZYME AND AN ENZYMEPROTECTING AGENT AND DETERGENT COMPOSITIONS CONTAINING SUCH GRANULES”which application is incorporated herein by reference in its entirety.Likewise, the granule can be formulated so as to contain materials toreduce the rate of dissolution of the granule into the wash medium. Suchmaterials and granules are disclosed in U.S. Pat. No. 5,254,283 which isincorporated herein by reference in its entirety.

The detergent compositions of this invention employ a surface activeagent, i.e., surfactant, including anionic, non-ionic and ampholyticsurfactants well known for their use in detergent compositions.

Suitable anionic surfactants for use in the detergent composition ofthis invention include linear or branched alkylbenzenesulfonates; alkylor alkenyl ether sulfates having linear or branched alkyl groups oralkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates;alkanesul-fonates and the like. Suitable counter ions for anionicsurfactants include alkali metal ions such as sodium and potassium;alkaline earth metal ions such as calcium and magnesium; ammonium ion;and alkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants include quaternary ammonium salt sulfonates,betaine-type ampholytic surfactants, and the like. Such ampholyticsurfactants have both the positive and negative charged groups in thesame molecule.

Nonionic surfactants generally comprise polyoxyal-kylene ethers, as wellas higher fatty acid alkanolamides or alkylene oxide adduct thereof,fatty acid glycerine monoesters, and the like.

Suitable surfactants for use in this invention are disclosed in BritishPatent Application No. 2 094 826 A, the disclosure of which isincorporated herein by reference.

Mixtures of such surfactants can also be used.

The surfactant or a mixture of surfactants is generally employed in thedetergent compositions of this invention in an amount from about 1weight percent to about 95 weight percent of the total detergentcomposition and preferably from about 5 weight percent to about 45weight percent of the total detergent composition. Upon dilution in thewash medium, the surfactant concentration is generally about 500 ppm ormore; and preferably, from about 1000 ppm to 15,000 ppm.

In addition to the cellulase composition and the surfactant(s), thedetergent compositions of this invention can optionally contain one ormore of the following components:

Hydrolases Except Cellulase

Such hydrolases include carboxylate ester hydrolase, thioesterhydrolase, phosphate monoester hydrolase, and phosphate diesterhydrolase which act on the ester bond; glycoside hydrolase which acts onglycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds;thioether hydrolase which acts on the ether bond; andα-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase,acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptidehydrolase which act on the peptide bond. Preferable among them arecarboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptidehydrolase. Suitable hydrolases include (1) proteases belonging topeptidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin,chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C,papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin,aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein,gastrisin, cathepsin D., bromelin, keratinase, chymotrypsin C, pepsin C,aspergillopeptidase B, urokinase, carboxypeptidase A and B, andaminopeptidase; (2) glycoside hydrolases (cellulase which is anessential ingredient is excluded from this group) α-amylase, β-amylase,gluco amylase, invertase, lysozyme, pectinase, chitinase, anddextranase. Preferably among them are α-amylase and β-amylase. Theyfunction in acid to neutral systems, but one which is obtained frombacteria exhibits high activity in an alkaline system; (3) carboxylateester hydrolase including carboxyl esterase, lipase, pectin esterase,and chlorophyllase. Especially effective among them is lipase.

Trade names of commercial products and producers are as follows:“Alkalase”, “Esperase”, “Savinase”, “AMG”, “BAN”, “Fungamill”,“Sweetzyme”, “Thermamyl” (Novo Industry, Copenhagen, Denmark);“Maksatase”, “High-alkaline protease”, “Amylase THC”, “Lipase” (GistBrocades, N. V., Delft, Holland); “Protease B-400”, “Protease B-4000”,“Protease AP”, “Protease AP 2100” (Schweizerische Ferment A. G., Basel,Switzerland); “CRD Protease” (Monsanto Company, St. Louis, Mo.);“Piocase” (Piopin Corporation, Monticello, Ill.); “Pronase P”, “PronaseAS”, “Pronase AF” (Kaken Chemical Co., Ltd., Japan); “Lapidase P-2000”(Lapidas, Secran, France); protease products (Tyler standard sieve, 100%pass 16 mesh and 100% on 150 mesh) (Clington Corn Products, Division ofStandard Brands Corp., New York); “Takamine”, “Bromelain 1:10”, “HTProtease 200”, “Enzyme L-W” (obtained from fungi, not from bacteria)(Miles Chemical Company, Elkhart, Ind.); “Rhozyme P-11 Conc.”,“Pectinol”, “Lipase B”, “Rhozyme PF”, “Rhozyme J-25” (Rohm & Haas,Genencor, South San Francisco, Calif.); “Ambrozyme 200” (Jack Wolf &Co., Ltd., Subsidiary of Nopco Chemical Company, Newark, N.J.); “ATP40”, “ATP 120”, “ATP 160” (Lapidas, Secran, France); “Oripase” (Nagase &Co., Ltd., Japan).

The hydrolase other than cellulase is incorporated into the detergentcomposition as much as required according to the purpose. It shouldpreferably be incorporated in an amount of 0.001 to 5 weight percent,and more preferably 0.02 to 3 weight percent, in terms of purified one.This enzyme should be used in the form of granules made of crude enzymealone or in combination with other components in the detergentcomposition. Granules of crude enzyme are used in such an amount thatthe purified enzyme is 0.001 to 50 weight percent in the granules. Thegranules are used in an amount of 0.002 to 20 and preferably 0.1 to 10weight percent. As with cellulases, these granules can be formulated soas to contain an enzyme protecting agent and a dissolution retardantmaterial.

Cationic Surfactants and Long-chain Fatty Acid Salts

Such cationic surfactants and long-chain fatty acid salts includesaturated or unsaturated fatty acid salts, alkyl or alkenyl ethercarboxylic acid salts, α-sulfofatty acid salts or esters, aminoacid-type surfactants, phosphate ester surfactants, quaternary ammoniumsalts including those having 3 to 4 alkyl substituents and up to 1phenyl substituted alkyl substituents. Suitable cationic surfactants andlong-chain fatty acid salts are disclosed in British Patent ApplicationNo. 2 094 826 A, the disclosure of which is incorporated herein byreference. The composition may contain from about 1 to about 20 weightpercent of such cationic surfactants and long-chain fatty acid salts.

Builders

A. Divalent Sequestering Agents

The composition may contain from about 0 to about 50 weight percent ofone or more builder components selected from the group consisting ofalkali metal salts and alkanolamine salts of the following compounds:phosphates, phosphonates, phosphonocarboxylates, salts of amino acids,aminopolyacetates high molecular electrolytes, non-dissociatingpolymers, salts of dicarboxylic acids, and aluminosilicate salts.Suitable divalent sequestering gents are disclosed in British PatentApplication No. 2 094 826 A, the disclosure of which is incorporatedherein by reference.

B. Alkalis or Inorganic Electrolytes

The composition may contain from about 1 to about 50 weight percent,preferably from about 5 to about 30 weight percent, based on thecomposition of one or more alkali metal salts of the following compoundsas the alkalis or inorganic electrolytes: silicates, carbonates andsulfates as well as organic alkalis such as triethanolamine,diethanolamine, monoethanolamine and triisopropanolamine.

Antiredeposition Agents

The composition may contain from about 0.1 to about 5 weight percent ofone or more of the following compounds as antiredeposition agents:polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone andcarboxymethylcellulose.

Among them, a combination of carboxymethyl-cellulose and/or polyethyleneglycol with the cellulase composition of the present invention providesfor an especially useful dirt removing composition.

Bleaching Agents

The use of the cellulase of the present invention in combination with ableaching agent such as sodium percarbonate, sodium perborate, sodiumsulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxideadduct or/and a photo-sensitive bleaching dye such as zinc or aluminumsalt of sulfonated phthalocyanine further improves the detergentingeffects.

Bluing Agents and Fluorescent Dyes

Various bluing agents and fluorescent dyes may be incorporated in thecomposition, if necessary. Suitable bluing agents and fluorescent dyesare disclosed in British Patent Application No. 2 094 826 A, thedisclosure of which is incorporated herein by reference.

Caking Inhibitors

The following caking inhibitors may be incorporated in the powderydetergent: p-toluenesulfonic acid salts, xylenesulfonic acid salts,acetic acid salts, sulfosuccinic acid salts, talc, finely pulverizedsilica, clay, calcium silicate (such as Micro-Cell of Johns ManvilleCo.), calcium carbonate and magnesium oxide.

Masking Agents for Factors Inhibiting the Cellulase Activity

The cellulase composition of this invention are deactivated in somecases in the presence of copper, zinc, chromium, mercury, lead,manganese or silver ions or their compounds. Various metal chelatingagents and metal-precipitating agents are effective against theseinhibitors. They include, for example, divalent metal ion sequesteringagents as listed in the above item with reference to optional additivesas well as magnesium silicate and magnesium sulfate.

Cellobiose, glucose and gluconolactone act sometimes as the inhibitors.It is preferred to avoid the co-presence of these saccharides with thecellulase as far as possible. In case the co-presence in unavoidable, itis necessary to avoid the direct contact of the saccharides with thecellulase by, for example, coating them.

Long-chain-fatty acid salts and cationic surfactants act as theinhibitors in some cases. However, the co-presence of these substanceswith the cellulase is allowable if the direct contact of them isprevented by some means such as tableting or coating.

The above-mentioned masking agents and methods may be employed, ifnecessary, in the present invention.

Cellulase-Activators

The activators vary depending on variety of the cellulases. In thepresence of proteins, cobalt and its salts, magnesium and its salts, andcalcium and its salts, potassium and its salts, sodium and its salts ormonosac-charides such as mannose and xylose, the cellulases areactivated and their deterging powers are improved remarkably.

Antioxidants

The antioxidants include, for example, tert-butyl-hydroxytoluene,4,4′-butylidenebis(6-tert-butyl-3-methylphenol),2,2′-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol,distyrenated cresol, monostyrenated phenol, distyrenated phenol and1,1-bis(4-hydroxy-phenyl)cyclohexane.

Solubilizers

The solubilizers include, for example, lower alcohols such as ethanol,benzenesulfonate salts, lower alkylbenzenesulfonate salts such asp-toluenesulfonate salts, glycols such as propylene glycol,acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acidamides, benzoate salts and urea.

The detergent composition of the present invention can be used in abroad pH range of from acidic to alkaline pH. In a preferred embodiment,the detergent composition of the present invention can be used inalkaline detergent wash media and more preferably, alkaline detergentwash media having a pH of from above 7 to no more than about 11.

Aside from the above ingredients, perfumes, buffers, preservatives, dyesand the like can be used, if desired, with the detergent compositions ofthis invention. Such components are conventionally employed in amountsheretofore used in the art.

When a detergent base used in the present invention is in the form of apowder, it may be one which is prepared by any known preparation methodsincluding a spray-drying method and a granulation method. The detergentbase obtained particularly by the spray-drying method and/orspray-drying granulation method are preferred. The detergent baseobtained by the spray-drying method is not restricted with respect topreparation conditions. The detergent base obtained by the spray-dryingmethod is hollow granules which are obtained by spraying an aqueousslurry of heat-resistant ingredients, such as surface active agents andbuilders, into a hot space. The granules have a size of from 50 to 2000micrometers. After the spray-drying, perfumes, enzymes, bleachingagents, inorganic alkaline builders may be added. With a highly dense,granular detergent base obtained such as by the spray-drying-granulationmethod, various ingredients may also be added after the preparation ofthe base.

When the detergent base is a liquid, it may be either a homogeneoussolution or an inhomogeneous dispersion. For removing the decompositionof carboxymethylcellulose by the cellulase in the detergent, it isdesirable that carboxymethylcellulose is granulated or coated before theincorporation in the composition.

The detergent compositions of this invention may be incubated withcellulose containing fabric, for example soiled fabrics, in industrialand household uses at temperatures, reaction times and liquor ratiosconventionally employed in these environments. The incubationconditions, i.e., the conditions effective for treating cellulosecontaining fabrics with detergent compositions according to the presentinvention, will be readily ascertainable by those of skill in the art.Accordingly, the appropriate conditions effective for treatment with thepresent detergents will correspond to those using similar detergentcompositions which include wild-type cellulase.

As indicated above, detergents according to the present invention mayadditionally be formulated as a pre-wash in the appropriate solution atan intermediate pH where sufficient activity exists to provide desiredimprovements in abrasion and reduced strength loss. When the detergentcomposition is a pre-soak (e.g., pre-wash or pre-treatment) composition,either as a liquid, spray, gel or paste composition, the truncatedcellulase enzyme is generally employed from about 0.0001 to about 1weight percent based on the total weight of the pre-soak orpre-treatment composition. In such compositions, a surfactant mayoptionally be employed and when employed, is generally present at aconcentration of from about 0.005 to about 20 weight percent based onthe total weight of the pre-soak. The remainder of the compositioncomprises conventional components used in the pre-soak, i.e., diluent,buffers, other enzymes (proteases), and the like at their conventionalconcentrations.

Also, it is contemplated that compositions comprising truncatedcellulase enzymes described herein can be used in home use as a standalone composition suitable for restoring color to faded fabrics (see,for example, U.S. Pat. No. 4,738,682, which is incorporated herein byreference in its entirety) as well as used in a spot-remover.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given with theunderstanding that they are being offered to illustrate the presentinvention and should not be construed in any way as limiting its scope.

EXAMPLES Example 1 Cloning and Expression of EGI Core Domain Using itsOwn Promoter, Terminator and Signal Sequence

Part 1. Cloning

The complete egl1 gene used in the construction of the EG1 core domainexpression plasmid, PEG1Δ3′ pyr, was obtained from the plasmidPUC218::EG1. (See FIG. 6) The 3′ terminator region of egl1 was ligatedinto PUC218 (Korman, D. et al Curr Genet 17:203-212, 1990) as a 300 bpBsmI-EcoRI fragment along with a synthetic linker designed to replacethe 3′ intron and cellulose binding domain with a stop codon andcontinue with the egl1 terminator sequences. The resultant plasmid,PEG1T, was digested with HindIII and BsmI and the vector fragment wasisolated from the digest by agarose gel electrophoresis followed byelectroelution. The egl1 gene promoter sequence and core domain of egl1were isolated from PUC218::EG1 as a 2.3 kb HindIII-SstI fragment andligated with the same synthetic linker fragment and the HindIII-BsmIdigested PEG1T to form PEG1Δ3′.

The net result of these operations is to replace the 3′ intron andcellulose binding domain of egl1 with synthetic oligonucleotides of 53and 55 bp. These place a TAG stop codon after serine 415 and thereaftercontinued with the egl1 terminator up to the BsmI site.

Next, the T. longibrachiatum selectable marker, pyr4, was obtained froma previous clone p219M (Smith et al 1991), as an isolated 1.6 kbEcoRI-HindIII fragment. This was incorporated into the final expressionplasmid, PEG1Δ3′ pyr, in a three way ligation with PUC18 plasmiddigested with EcoRI and dephosphorylated using calf alkaline phosphataseand a HindIII-EcoRI fragment containing the egl1 core domain fromPEG1Δ3′.

Part 2. Transformation and Expression

A large scale DNA prep was made of PEG1Δ3′ pyr and from this the EcoRIfragment containing the egl1 core domain and pyr4 gene was isolated bypreparative gel electrophoresis. The isolated fragment was transformedinto the uridine auxotroph version of the quad deleted strain, 1A52pyr13 (described in U.S. patent application Ser. Nos. 07/770,049,08/048,728 and 08/048,881, incorporated by reference in its entiretyherein), and stable transformants were identified.

To select which transformants expressed egl1 core domain thetransformants were grown up in shake flasks under conditions thatfavored induction of the cellulase genes (Vogels+1% lactose). After 4-5days of growth, protein from the supernatants was concentrated andeither 1) run on SDS polyacrylamide gels prior to detection of the egl1core domain by Western analysis using EGI polyclonal antibodies or 2)the concentrated supernatants were assayed directly using REB carboxymethyl cellulose as an endoglucanase specific substrate and the resultscompared to the parental strain 1A52 as a control. Transformantcandidates were identified as possibly producing a truncated EGI coredomain protein. Genomic DNA and total mRNA was isolated from thesestrains following growth on Vogels+1% lactose and Southern and Northernblot experiments performed using an isolated DNA fragment containingonly the egl1 core domain. These experiments demonstrated thattransformants could be isolated having a copy of the egl1 core domainexpression cassette integrated into the genome of 1A52 and that thesesame transformants produced egl1 core domain mRNA.

One transformant was then grown using media suitable for cellulaseproduction in Trichoderma well known in the art that was supplementedwith lactose (Warzymoda, M. et al 1984 French Patent No. 2555603) in a14 L fermentor. The resultant broth was concentrated and the proteinscontained therein were separated by SDS polyacrylamide gelelectrophoresis and the Egl1 core domain protein identified by Westernanalysis. (See Example 3 below). It was subsequently estimated that theprotein concentration of the fermentation supernatant was about 5-6 g/Lof which approximately 1.7-4.4 g/L was EGI core domain based on CMCaseactivity. This value is based on an average of several EGI corefermentations that were performed.

In a similar manner, any other cellulase domain or derivative thereofmay be produced by procedures similar to those discussed above.

Example 2 Purification of EGI and EGII Catalytic Cores

Part 1. EGI Catalytic Core

The EGI core was purified in the following manner. The concentrated (UF)broth was diluted to 14 mg/ml in 23 mM Na Acetate pH 5.0. Two hundredgrams of avicel cellulose gel (FMC Bioproducts, Type PH-101) was addedto the diluted EGI core broth and mixed at room temperature for fortyfive minutes. The avicel was removed from the broth by centrifugation,resulting in an enriched EGI core solution. This solution was thenbuffer exchanged into 10 mM TES pH 7.5 using a Amicon stirred cellconcentrator with a PM 10 membrane (diaflo ultra filtration membranes,Amicon Cat # 13132MEM 5468A). The EGI core sample was then loaded ontoan anion exchange column (Q-sepharose fast flow, Pharmacia Cat #17-0510-01) and eluted in a salt gradient from 0 to 0.5M NaCl in 10 mMTES pH 7.5. The fractions which contained the EGI core were combined andconcentrated using the Amicon stirred cell concentrator mentioned above.

Part 2. EGII Catalytic Core

EGII core was purified in the following manner. The concentrated (UF)broth was filtered using diatomaceous earth. Ammonium sulfate was addedto the broth to a final concentration of 1M (NH₄)₂SO₄ and this mixturewas then loaded on to a hydrophobic column (phenyl-sepharose fast flow,Pharmacia, cat #17-0965-01). The column was washed with 0.75M ammoniumsulfate before elution of the EGII core with 0.5M ammonium sulfate. Thefractions containing the core were pooled and concentrated using atangential flow ultra filtration membrane (Filtron MinisetteUltrafiltration System, cat #FS018K01) with an Omega 10K Membrane(Filtron, cat# FS010K75). 100 grams of avicel cellulose gel (FMCBioproducts, Type PH-101) was added for every gram of concentrated EGIIcore and mixed for 40 minutes. The avicel was removed by centrifugationresulting in an enriched EGII core solution.

Example 3 Cloning and Expression of CBHII Core Domain Using the CBHIPromoter, Terminator and Signal Sequence from CBHII

Part 1. Construction of the T. longibrachiatum General-purposeExpression Plasmid-PTEX

The plasmid, PTEX was constructed following the methods of Sambrook etal. (1989), supra, and is illustrated in FIG. 7. This plasmid has beendesigned as a multi-purpose expression vector for use in the filamentousfungus Trichoderma longibrachiatum. The expression cassette has severalunique features that make it useful for this function. Transcription isregulated using the strong CBH I gene promoter and terminator sequencesfor T. longibrachiatum. Between the CBHI promoter and terminator thereare unique PmeI and SstI restriction sites that are used to insert thegene to be expressed. The T. longibrachiatum pyr4 selectable marker genehas been inserted into the CBHI terminator and the whole expressioncassette (CBHI promoter-insertion sites-CBHI terminator-pyr4 gene-CBHIterminator) can be excised utilizing the unique NotI restriction site orthe unique NotI and NheI restriction sites.

This vector is based on the bacterial vector, pSL1180 (Pharmacia Inc.,Piscataway, N.J.), which is a PUC-type vector with an extended multiplecloning site. One skilled in the art would be able to construct thisvector based on the flow diagram illustrated in FIG. 7. (See also U.S.patent application Ser. No. 07/954,113 filed Sep. 30, 1992 entitled“Stonewashing of Denim Garments Using Endoglucanase I & III” for theconstruction of PTEX expression plasmid.)

It would be possible to construct plasmids similar to PTEX-truncatedcellulases or derivatives thereof described in the present inventioncontaining any other piece of DNA sequence replacing the truncatedcellulase gene.

Part 2. Cloning

The complete cbh2 gene used in the construction of the CBHII core domainexpression plasmid, PTEX CBHII core, was obtained from the plasmidPUC219::CBHII (Korman, D. et al, 1990, Curr Genet 17:203-212). Thecellulose binding domain, positioned at the 5′ end of the cbh2 gene, isconveniently located between an XbaI and SnaBI restriction sites. Inorder to utilize the XbaI site an additional XbaI site in the polylinkerwas destroyed. PUC219::CBHII was partially digested with XbaI such thatthe majority of the product was linear. The XbaI overhangs were filledin using T4 DNA polymerase and ligated together under conditionsfavoring self ligation of the plasmid. This has the effect of destroyingthe blunted site which, in 50% of the plasmids, was the XbaI site in thepolylinker. Such a plasmid was identified and digested with XbaI andSnaBI to release the cellulose binding domain. The vector-CBHII coredomain was isolated and ligated with the following syntheticoligonucleotides designed to join the XbaI site with the SnaBI site atthe signal peptidase cleavage site and papain cleavage point in thelinker domain.

   XbaI                          SnaBI 5′ CTA GAG CGG TCG GGA ACC GCT AC3′ (SEQ ID NO:42)    3′   TC CTC GCC AGC CCT TGG CGA TG 5′ (SEQ ID   Leu Glu Glu Arg Ser Gly Thr Ala Thr NO:43)

The resultant plasmid, pUCΔCBD CBHII, was digested with NheI and theends blunted by incubation with T4 DNA polymerase and dNTPs. After whichthe linear blunted plasmid DNA was digested with BglII and the Nhe(blunt) BglII fragment containing the CBHII signal sequence and coredomain was isolated.

The final expression plasmid was engineered by digesting the generalpurpose expression plasmid, pTEX (disclosed in U.S. patent applicationSer. No. 07/954,113, incorporated in its entirety by references, anddescribed in Part 3 below), with SstII and PmeI and ligating the CBHIINheI (blunt)-BglII fragment from pUCΔCBD CBHII downstream of the cbh1promoter using a synthetic oligonucleotide having the sequence CGCTAG tofill in the BglII overhang with the SstII overhang. The construction ofthe resulting PTEC CBHIIcore expression vector is summarized in FIG. 9.

The pTEX-CBHI core expression plasmid was prepared in a similar manneras PTEX-CBHII core described in the above example. Its construction isexemplified in FIG. 8.

Part 3. Transformation and Expression

A large scale DNA prep was made of pTEX CBHIIcore and from this the NotIfragment containing the CBHII core domain under the control of the cbh1transcriptional elements and pyr4 gene was isolated by preparative gelelectrophoresis. The isolated fragment was transformed into the uridineauxotroph version of the quad deleted strain, 1A52 pyr13, and stabletransformants were identified.

To select which transformants expressed cbh2 core domain genomic DNA wasisolated from strains following growth on Vogels+1% glucose and Southernblot experiments performed using an isolated DNA fragment containingonly the cbh2 core domain. Transformants were isolated having a copy ofthe cbh2 core domain expression cassette integrated into the genome of1A52. Total mRNA was isolated from the two strains following growth for1 day on Vogels+1% lactose. The mRNA was subjected to Northern analysisusing the cbh2 coding region as a probe. Transformants expressing cbh2core domain mRNA were identified.

Two transformants were grown under the same conditions as previouslydescribed in Example 1 in 14L fermentors. The resultant broth wasconcentrated and the proteins contained therein were separated by SDSpolyacrylamide gel electrophoresis and the CBHII core domain proteinidentified by Western analysis. One transformant, #15, produced aprotein of the correct size and reactivity to CBHII polyclonalantibodies.

It was subsequently estimated that the protein concentration of thefermentation supernatant after purification was 10 g/L of which 30-50%was CBHII core domain (See Example 4).

One may obtain any other novel truncated cellulase core domain proteinor derivative thereof by employing the methods described above.

Example 4 Purification of CBHI and CBHII Catalytic Cores

Part 1. CBHI Catalytic Core

The CBHI core was purified from broth in the following manner. The CBHIcore ultrafiltered (UF) broth was filtered using diatomaceous earth anddiluted in 10 mM TES pH 6.8 to a conductivity of 1.5 mOhm. The dilutedCBHI core was then loaded onto an anion exchange column (Q-Sepharosefast flow, Pharmacia cat # 17-0510-01) equilibrated in 10 mM TES pH 6.8.The CBHI core was separated from the majority of the other proteins inthe broth using a gradient elution in 10 mM TES pH 6.8 from 0 to 1MNaCl. The fractions containing the CBHI core were then concentrated onan Amicon stirred cell concentrator with a PM 10 membrane (diaflo ultrafiltration membranes, Amicon Cat # 13132MEM 5468A). This stepconcentrated the core, and additionally effected a separation of thecore from lower molecular weight proteins. The resulting fractions weregreater than 85% pure CBHI core. The purest fraction was sequenceverified to be the CBHI core.

Part 2. CBHII Catalytic Core

It is predicted that CBHII catalytic core will purify in a mannersimilar to that of CBHII cellulase because of its similar biochemicalproperties. The theoretical pI of the CBHII core is less than half a pHunit lower than that of CBHII. Additionally, CBHII catalytic core isapproximately 80% of the molecular weight of CBHII. Therefore, thefollowing proposed purification protocol is based on the purificationmethod used for CBHII. The diatomaceous earth treated, ultra filtered(UF) CBHII core broth is diluted into 10 mM TES pH 6.8 to a conductivityof <0.7 mOhm. The diluted CBHII core is then loaded onto an anionexchange column (Q-Sepharose fast flow, Pharmacia, cat # 17 0510-01)equilibrated in 10 mM TES pH 6.8. A salt gradient from 0 to 1M NaCl in10 mM TES pH 6.8 is used to elute the CBHII core off the column. Thefractions which contain the CBHII core is then buffer exchanged into 2mM sodium succinate buffer and loaded onto a cation exchange column(SP-sephadex C-50). The CBHII core is next eluted from the column with asalt gradient from 0 to 100 mM NaCl.

Example 5 Stonewashing with Truncated Cellulase Enzyme

This example demonstrates that the use of truncated cellulase catalyticcores, and in particular truncated endoglucanase catalytic cores,surprisingly results in a reduced redeposition of dye onto a cellulosecontaining (denim) fabric during stonewashing at an equivalent level ofabrasion to non-truncated cellulase. Further, the combination oftruncated CBH-type catalytic core and non-truncated EG-type cellulaseresulted in significantly improved backstaining characteristics whencompared to non-truncated combinations at equivalent levels of abrasion.Denim is cotton cloth which has been dyed. Methods for imparting a stonewashed appearance to denim fabrics are described in U.S. Pat. No.4,832,864 and U.S. Ser. No. 07/954,113 which are incorporated herein byreference in its entirety.

Truncated cellulase catalytic cores and cellulase components werepurified for use in this example as follows:

a) EGIII cellulase component was purified according to the methoddescribed in U.S. Pat. No. 5,328,841 which disclosure is incorporatedherein by reference in its entirety.

b) truncated EGI catalytic cellulase core was purified by the method ofExample 2.

c) truncated EGII catalytic cellulase core was purified by the method ofExample 2.

d) truncated CBHI catalytic core was prepared according to the method ofExample 4.

e) EGI cellulase component was purified for use in stonewashingapplications in the following manner. The concentrated (UF) broth wasdiluted in 25 mM sodium acetate buffer pH 5.0 to a concentration of 7mg/ml protein. 450 grams of avicel cellulose gel (FMC Bioproducts, TypePH-101) was added to the diluted broth and mixed at room temperature forthirty minutes. The avicel was removed from the broth by centrifugationresulting in an enriched EGI catalytic core.

f) EGII cellulase component was purified for use in stonewashing in thefollowing manner. The concentrated (UF) broth was filtered throughdiatomaceous earth and buffer exchanged into citrate-phosphate pH 7, 0.4mOhm using an ultrafiltration unit (Filtron Minisette UltrafiltrationSystem, cat # FS018K01) with an Omega 10K Membrane (Filtron, cat. #FS010K75). This material was then loaded onto an anion exchange column(Q-sepharose fast flow, Pharmacia, cat. #17-0510-01) equilibrated in theabove citrate-phosphate buffer. EGII was collected in the column flowthrough and fubber exchanged into sodium citrate pH 3.25, 0.5 mOhmsusing the ultrafiltration unit mentioned above. This material was thenloaded onto a cation exchange column (SP-Spherodex LS, IBF Biotechnics,cat #262041). EGII was then eluted off the column in a linear gradientfrom pH 3.25 to 5.4. The fractions containing EGII were pooled and thenused in stonewashing applications.

g) CBH I cellulase component was purified according to the methoddescribed in Example 15 of U.S. patent application Ser. No. 07/954,113.

These truncated cellulase catalytic core domains and cellulasecomponents were tested alone or in combination for their ability toimpart a stonewashed appearance to dyed denims. Specifically, thestonewashed denim fabrics were prepared using an industrial washer anddryer under the following conditions:

Citrate/phosphate buffer@ pH 5

38L total volume

120-135° F.

Six pair of denim pants with an equal weight of ballast

1 hour run time

15-30 ppm EGI, EGII, EGIII; 50 ppm CBH I and CBHI catalytic core; and30-70 ppm EGI catalytic core and EGII catalytic core.

A detergent post wash included 90 grams of AATCC detergent withoutbrighteners in the case of CBHI, CBHI catalytic core, EGIII plus CBHIand EGIII plus CBHI catalytic core.

The fabrics were evaluated for their stonewashed appearance (abrasion)and also for redeposition of dye onto the fabric by five panelists. Thefabrics were graded using a scale consisting of denim swatches whichhave incrementally increasing levels of abrasion or redeposition. Thescales are numerated from 1-10; 1 representing the jeans with the leastamount of abrasion or redeposition (desize denim), and 10 representing ahighly abraded or redeposited denim. The appearance of the treated jeanswas directly compared to the denim swatches in the scale and give anumber based on the swatch in the scale they most closely matched. Theresults of the evaluation are shown in FIGS. 10 and 11.

As shown in FIG. 10, truncated EGI catalytic core and truncated EGIIcatalytic core provide significant advantages over their un-modified,native state counterparts in abrasion and reduced redeposition.

As shown in FIG. 11, the addition of CBHI cellulase component to EGIIIresulted in a slight enhancement of stonewashing but a large increase inbackstaining (redeposition) of dye onto the fabric. However, theaddition of truncated CBHI catalytic core to EGIII solution improvedresults in both increased abrasion and decreased redeposition. In fact,the addition of truncated CBHI catalytic core to EGIII cellulasedecreased redeposition to a level below that of EGIII alone.

As these result show, the use of truncated cellulase core duringtreatment of cellulose containing fabrics surprisingly results indecreased backstaining while either maintaining an equivalent orsuperior level of abrasion. Further, this result is achieved either whenthe truncated cellulase is used alone, or when it is mixed with anotherwild-type or truncated cellulase. These results are both unexpected andsurprising.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 43 <210> SEQ ID NO 1 <211> LENGTH: 93<212> TYPE: DNA <213> ORGANISM: Trichoderma longibrachiatum<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(93)<400> SEQUENCE: 1 ggc cag tgc ggc ggt att ggc tac agc ggc cc#c acg gtc tgc gcc agc       48Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pr #o Thr Val Cys Ala Ser 1               5   #                 10  #                 15ggc aca act tgc cag gtc ctg aac cct tac ta #c tct cag tgc ctg           #93 Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Ty #r Ser Gln Cys Leu             20      #             25      #             30<210> SEQ ID NO 2 <211> LENGTH: 31 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 2Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pr #o Thr Val Cys Ala Ser 1               5   #                10   #                15Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Ty #r Ser Gln Cys Leu            20       #            25       #            30<210> SEQ ID NO 3 <211> LENGTH: 166 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(20) <221> NAME/KEY: CDS<222> LOCATION: (70)...(166) <400> SEQUENCE: 3caa gct tgc tca agc gtc tg gtaattatgt gaaccctctc# aagagaccca           50 Gln Ala Cys Ser Ser Val Trp  1               5aatactgaga tatgtcaag g ggc caa tgt ggt ggc cag #aat tgg tcg ggt       100                   #     Gly Gln Cys Gly Gly Gln Asn Tr #p Ser Gly                   #              10     #              15ccg act tgc tgt gct tcc gga agc aca tgc gt#c tac tcc aac gac tat      148Pro Thr Cys Cys Ala Ser Gly Ser Thr Cys Va #l Tyr Ser Asn Asp Tyr         20          #         25          #         30tac tcc cag tgt ctt ccc          #                   #                  # 166 Tyr Ser Gln Cys Leu Pro      35 <210> SEQ ID NO 4 <211> LENGTH: 39<212> TYPE: PRT <213> ORGANISM: Trichoderma longibrachiatum<400> SEQUENCE: 4 Gln Ala Cys Ser Ser Val Trp Gly Gln Cys Gl#y Gly Gln Asn Trp Ser  1               5   #                10  #                15 Gly Pro Thr Cys Cys Ala Ser Gly Ser Thr Cy#s Val Tyr Ser Asn Asp             20       #            25      #            30 Tyr Tyr Ser Gln Cys Leu Pro         35 <210> SEQ ID NO 5<211> LENGTH: 156 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(82) <221> NAME/KEY: CDS<222> LOCATION: (140)...(156) <400> SEQUENCE: 5cac tgg ggg cag tgc ggt ggc att ggg tac ag#c ggg tgc aag acg tgc       48His Trp Gly Gln Cys Gly Gly Ile Gly Tyr Se #r Gly Cys Lys Thr Cys 1               5   #                 10  #                 15acg tcg ggc act acg tgc cag tat agc aac ga #c t gttcgtatcc             # 92 Thr Ser Gly Thr Thr Cys Gln Tyr Ser Asn As #p              20     #             25 ccatgcctga cgggagtgat tttgagatgc taaccgctaa aatacag ac #tac tcg       147                    #                  #               Tyr Tyr  #Ser                    #                  #                   #      30 caa tgc ctt              #                   #                   #        156 Gln Cys Leu<210> SEQ ID NO 6 <211> LENGTH: 33 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 6His Trp Gly Gln Cys Gly Gly Ile Gly Tyr Se #r Gly Cys Lys Thr Cys 1               5   #                10   #                15Thr Ser Gly Thr Thr Cys Gln Tyr Ser Asn As #p Tyr Tyr Ser Gln Cys            20       #            25       #            30 Leu<210> SEQ ID NO 7 <211> LENGTH: 108 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(108) <400> SEQUENCE: 7cag cag act gtc tgg ggc cag tgt gga ggt at#t ggt tgg agc gga cct       48Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Il #e Gly Trp Ser Gly Pro 1               5   #                 10  #                 15acg aat tgt gct cct ggc tca gct tgt tcg ac#c ctc aat cct tat tat       96Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Th #r Leu Asn Pro Tyr Tyr             20      #             25      #             30gcg caa tgt att             #                   #                  #      108 Ala Gln Cys Ile          35 <210> SEQ ID NO 8<211> LENGTH: 36 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 8Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Il #e Gly Trp Ser Gly Pro 1               5   #                10   #                15Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Th #r Leu Asn Pro Tyr Tyr            20       #            25       #            30Ala Gln Cys Ile         35 <210> SEQ ID NO 9 <211> LENGTH: 1453<212> TYPE: DNA <213> ORGANISM: Trichoderma longibrachiatum<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(410)<221> NAME/KEY: CDS <222> LOCATION: (478)...(1174) <221> NAME/KEY: CDS<222> LOCATION: (1238)...(1453) <400> SEQUENCE: 9cag tcg gcc tgc act ctc caa tcg gag act ca#c ccg cct ctg aca tgg       48Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr Hi #s Pro Pro Leu Thr Trp 1               5   #                 10  #                 15cag aaa tgc tcg tct ggt ggc act tgc act ca#a cag aca ggc tcc gtg       96Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gl #n Gln Thr Gly Ser Val             20      #             25      #             30gtc atc gac gcc aac tgg cgc tgg act cac gc#t acg aac agc agc acg      144Val Ile Asp Ala Asn Trp Arg Trp Thr His Al #a Thr Asn Ser Ser Thr         35          #         40          #         45aac tgc tac gat ggc aac act tgg agc tcg ac#c cta tgt cct gac aac      192Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Th #r Leu Cys Pro Asp Asn     50              #     55              #     60gag acc tgc gcg aag aac tgc tgt ctg gac gg#t gcc gcc tac gcg tcc      240Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gl #y Ala Ala Tyr Ala Ser 65                  # 70                  # 75                  # 80acg tac gga gtt acc acg agc ggt aac agc ct#c tcc att ggc ttt gtc      288Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Le #u Ser Ile Gly Phe Val                 85  #                 90  #                 95acc cag tct gcg cag aag aac gtt ggc gct cg#c ctt tac ctt atg gcg      336Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Ar #g Leu Tyr Leu Met Ala            100       #           105       #           110agc gac acg acc tac cag gaa ttc acc ctg ct#t ggc aac gag ttc tct      384Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Le #u Gly Asn Glu Phe Ser        115           #       120           #       125ttc gat gtt gat gtt tcg cag ctg cc gtaagtgact# taccatgaac             430 Phe Asp Val Asp Val Ser Gln Leu Pro    130               #   135ccctgacgta tcttcttgtg ggctcccagc tgactggcca atttaag g #tgc ggc ttg    487                    #                  #                   #Cys Gly Leu                    #                  #                   #        140aac gga gct ctc tac ttc gtg tcc atg gac gc#g gat ggt ggc gtg agc      535Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Al #a Asp Gly Gly Val Ser                145   #               150   #               155aag tat ccc acc aac acc gct ggc gcc aag ta#c ggc acg ggg tac tgt      583Lys Tyr Pro Thr Asn Thr Ala Gly Ala Lys Ty #r Gly Thr Gly Tyr Cys            160       #           165       #           170gac agc cag tgt ccc cgc gat ctg aag ttc at#c aat ggc cag gcc aac      631Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Il #e Asn Gly Gln Ala Asn        175           #       180           #       185gtt gag ggc tgg gag ccg tca tcc aac aac gc#a aac acg ggc att gga      679Val Glu Gly Trp Glu Pro Ser Ser Asn Asn Al #a Asn Thr Gly Ile Gly    190               #   195               #   200gga cac gga agc tgc tgc tct gag atg gat at#c tgg gag gcc aac tcc      727Gly His Gly Ser Cys Cys Ser Glu Met Asp Il #e Trp Glu Ala Asn Ser205                 2 #10                 2 #15                 2 #20atc tcc gag gct ctt acc ccc cac cct tgc ac#g act gtc ggc cag gag      775Ile Ser Glu Ala Leu Thr Pro His Pro Cys Th #r Thr Val Gly Gln Glu                225   #               230   #               235atc tgc gag ggt gat ggg tgc ggc gga act ta#c tcc gat aac aga tat      823Ile Cys Glu Gly Asp Gly Cys Gly Gly Thr Ty #r Ser Asp Asn Arg Tyr            240       #           245       #           250ggc ggc act tgc gat ccc gat ggc tgc gac tg#g aac cca tac cgc ctg      871Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp Tr #p Asn Pro Tyr Arg Leu        255           #       260           #       265ggc aac acc agc ttc tac ggc cct ggc tca ag#c ttt acc ctc gat acc      919Gly Asn Thr Ser Phe Tyr Gly Pro Gly Ser Se #r Phe Thr Leu Asp Thr    270               #   275               #   280acc aag aaa ttg acc gtt gtc acc cag ttc ga#g acg tcg ggt gcc atc      967Thr Lys Lys Leu Thr Val Val Thr Gln Phe Gl #u Thr Ser Gly Ala Ile285                 2 #90                 2 #95                 3 #00aac cga tac tat gtc cag aat ggc gtc act tt#c cag cag ccc aac gcc     1015Asn Arg Tyr Tyr Val Gln Asn Gly Val Thr Ph #e Gln Gln Pro Asn Ala                305   #               310   #               315gag ctt ggt agt tac tct ggc aac gag ctc aa#c gat gat tac tgc aca     1063Glu Leu Gly Ser Tyr Ser Gly Asn Glu Leu As #n Asp Asp Tyr Cys Thr            320       #           325       #           330gct gag gag gca gaa ttc ggc gga tcc tct tt#c tca gac aag ggc ggc     1111Ala Glu Glu Ala Glu Phe Gly Gly Ser Ser Ph #e Ser Asp Lys Gly Gly        335           #       340           #       345ctg act cag ttc aag aag gct acc tct ggc gg#c atg gtt ctg gtc atg     1159Leu Thr Gln Phe Lys Lys Ala Thr Ser Gly Gl #y Met Val Leu Val Met    350               #   355               #   360agt ctg tgg gat gat gtgagtttga tggacaaaca tgcgcgttg#a caaagagtca     1214 Ser Leu Trp Asp Asp 365agcagctgac tgagatgtta cag tac tac gcc aac atg ct#g tgg ctg gac tcc   1267                   #        Tyr Tyr Ala Asn Met Leu  #Trp Leu Asp Ser                   #        370          #        375acc tac ccg aca aac gag acc tcc tcc aca cc#c ggt gcc gtg cgc gga     1315Thr Tyr Pro Thr Asn Glu Thr Ser Ser Thr Pr #o Gly Ala Val Arg Gly380                 3 #85                 3 #90                 3 #95agc tgc tcc acc agc tcc ggt gtc cct gct ca#g gtc gaa tct cag tct     1363Ser Cys Ser Thr Ser Ser Gly Val Pro Ala Gl #n Val Glu Ser Gln Ser                400   #               405   #               410ccc aac gcc aag gtc acc ttc tcc aac atc aa#g ttc gga ccc att ggc     1411Pro Asn Ala Lys Val Thr Phe Ser Asn Ile Ly #s Phe Gly Pro Ile Gly            415       #           420       #           425agc acc ggc aac cct agc ggc ggc aac cct cc #c ggc gga aac             #1453 Ser Thr Gly Asn Pro Ser Gly Gly Asn Pro Pr #o Gly Gly Asn        430           #       435           #       440<210> SEQ ID NO 10 <211> LENGTH: 441 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 10Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr Hi #s Pro Pro Leu Thr Trp 1               5   #                10   #                15Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gl #n Gln Thr Gly Ser Val            20       #            25       #            30Val Ile Asp Ala Asn Trp Arg Trp Thr His Al #a Thr Asn Ser Ser Thr        35           #        40           #        45Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Th #r Leu Cys Pro Asp Asn    50               #    55               #    60Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gl #y Ala Ala Tyr Ala Ser65                   #70                   #75                   #80Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Le #u Ser Ile Gly Phe Val                85   #                90   #                95Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Ar #g Leu Tyr Leu Met Ala            100       #           105       #           110Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Le #u Gly Asn Glu Phe Ser        115           #       120           #       125Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gl #y Leu Asn Gly Ala Leu    130               #   135               #   140Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Va #l Ser Lys Tyr Pro Thr145                 1 #50                 1 #55                 1 #60Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Ty #r Cys Asp Ser Gln Cys                165   #               170   #               175Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Al #a Asn Val Glu Gly Trp            180       #           185       #           190Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Il #e Gly Gly His Gly Ser        195           #       200           #       205Cys Cys Ser Glu Met Asp Ile Trp Glu Ala As #n Ser Ile Ser Glu Ala    210               #   215               #   220Leu Thr Pro His Pro Cys Thr Thr Val Gly Gl #n Glu Ile Cys Glu Gly225                 2 #30                 2 #35                 2 #40Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Ar #g Tyr Gly Gly Thr Cys                245   #               250   #               255Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Ar #g Leu Gly Asn Thr Ser            260       #           265       #           270Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu As #p Thr Thr Lys Lys Leu        275           #       280           #       285Thr Val Val Thr Gln Phe Glu Thr Ser Gly Al #a Ile Asn Arg Tyr Tyr    290               #   295               #   300Val Gln Asn Gly Val Thr Phe Gln Gln Pro As #n Ala Glu Leu Gly Ser305                 3 #10                 3 #15                 3 #20Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cy #s Thr Ala Glu Glu Ala                325   #               330   #               335Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gl #y Gly Leu Thr Gln Phe            340       #           345       #           350Lys Lys Ala Thr Ser Gly Gly Met Val Leu Va #l Met Ser Leu Trp Asp        355           #       360           #       365Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Se #r Thr Tyr Pro Thr Asn    370               #   375               #   380Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gl #y Ser Cys Ser Thr Ser385                 3 #90                 3 #95                 4 #00Ser Gly Val Pro Ala Gln Val Glu Ser Gln Se #r Pro Asn Ala Lys Val                405   #               410   #               415Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gl #y Ser Thr Gly Asn Pro            420       #           425       #           430Ser Gly Gly Asn Pro Pro Gly Gly Asn         435           #       440<210> SEQ ID NO 11 <211> LENGTH: 1241 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(161) <221> NAME/KEY: CDS<222> LOCATION: (218)...(465) <221> NAME/KEY: CDS<222> LOCATION: (556)...(1241) <400> SEQUENCE: 11tcg gga acc gct acg tat tca ggc aac cct tt#t gtt ggg gtc act cct       48Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Ph #e Val Gly Val Thr Pro 1               5   #                 10  #                 15tgg gcc aat gca tat tac gcc tct gaa gtt ag#c agc ctc gct att cct       96Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Se #r Ser Leu Ala Ile Pro             20      #             25      #             30agc ttg act gga gcc atg gcc act gct gca gc#a gct gtc gca aag gtt      144Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Al #a Ala Val Ala Lys Val         35          #         40          #         45ccc tct ttt atg tgg ct gtaggtcctc ccggaaccaa ggc#aatctgt              191 Pro Ser Phe Met Trp Leu      50tactgaaggc tcatcattca ctgcag a gat act ctt gac a#ag acc cct ctc       242                    #             Asp Thr Leu #Asp Lys Thr Pro Leu                    #              55    #              60 atg gag caa acc ttg gcc gac atc cgc acc gc#c aac aag aat ggc ggt      290Met Glu Gln Thr Leu Ala Asp Ile Arg Thr Al #a Asn Lys Asn Gly Gly         65          #         70          #         75aac tat gcc gga cag ttt gtg gtg ata gac tt#g ccg gat cgc gat tgc      338Asn Tyr Ala Gly Gln Phe Val Val Ile Asp Le #u Pro Asp Arg Asp Cys     80              #     85              #     90gct gcc ctt gcc tcg aat ggc gaa tac tct at#t gcc gat ggt ggc gtc      386Ala Ala Leu Ala Ser Asn Gly Glu Tyr Ser Il #e Ala Asp Gly Gly Val 95                  #100                  #105                  #110gcc aaa tat aag aac tat atc gac acc att cg#t caa att gtc gtg gaa      434Ala Lys Tyr Lys Asn Tyr Ile Asp Thr Ile Ar #g Gln Ile Val Val Glu                115   #               120   #               125tat tcc gat atc cgg acc ctc ctg gtt att g #gtatgagttt aaacacctgc      485 Tyr Ser Asp Ile Arg Thr Leu Leu Val Ile            130       #           135ctcccccccc ccttcccttc ctttcccgcc ggcatcttgt cgttgtgcta ac#tattgttc    545 cctcttccag ag cct gac tct ctt gcc aac ctg gtg# acc aac ctc ggt act    596            Glu Pro Asp Ser #Leu Ala Asn Leu Val Thr Asn Leu Gly Thr                   #     140             #     145             #     150cca aag tgt gcc aat gct cag tca gcc tac ct#t gag tgc atc aac tac      644Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Le #u Glu Cys Ile Asn Tyr                155   #               160   #               165gcc gtc aca cag ctg aac ctt cca aat gtt gc#g atg tat ttg gac gct      692Ala Val Thr Gln Leu Asn Leu Pro Asn Val Al #a Met Tyr Leu Asp Ala            170       #           175       #           180ggc cat gca gga tgg ctt ggc tgg ccg gca aa#c caa gac ccg gcc gct      740Gly His Ala Gly Trp Leu Gly Trp Pro Ala As #n Gln Asp Pro Ala Ala        185           #       190           #       195cag cta ttt gca aat gtt tac aag aat gca tc#g tct ccg aga gct ctt      788Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Se #r Ser Pro Arg Ala Leu    200               #   205               #   210cgc gga ttg gca acc aat gtc gcc aac tac aa#c ggg tgg aac att acc      836Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr As #n Gly Trp Asn Ile Thr215                 2 #20                 2 #25                 2 #30agc ccc cca tcg tac acg caa ggc aac gct gt#c tac aac gag aag ctg      884Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Va #l Tyr Asn Glu Lys Leu                235   #               240   #               245tac atc cac gct att gga cct ctt ctt gcc aa#t cac ggc tgg tcc aac      932Tyr Ile His Ala Ile Gly Pro Leu Leu Ala As #n His Gly Trp Ser Asn            250       #           255       #           260gcc ttc ttc atc act gat caa ggt cga tcg gg#a aag cag cct acc gga      980Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gl #y Lys Gln Pro Thr Gly        265           #       270           #       275cag caa cag tgg gga gac tgg tgc aat gtg at#c ggc acc gga ttt ggt     1028Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Il #e Gly Thr Gly Phe Gly    280               #   285               #   290att cgc cca tcc gca aac act ggg gac tcg tt#g ctg gat tcg ttt gtc     1076Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Le #u Leu Asp Ser Phe Val295                 3 #00                 3 #05                 3 #10tgg gtc aag cca ggc ggc gag tgt gac ggc ac#c agc gac agc agt gcg     1124Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Th #r Ser Asp Ser Ser Ala                315   #               320   #               325cca cga ttt gac tcc cac tgt gcg ctc cca ga#t gcc ttg caa ccg gcg     1172Pro Arg Phe Asp Ser His Cys Ala Leu Pro As #p Ala Leu Gln Pro Ala            330       #           335       #           340cct caa gct ggt gct tgg ttc caa gcc tac tt#t gtg cag ctt ctc aca     1220Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Ph #e Val Gln Leu Leu Thr        345           #       350           #       355aac gca aac cca tcg ttc ctg        #                  #                1241 Asn Ala Asn Pro Ser Phe Leu     360              #   365 <210> SEQ ID NO 12 <211> LENGTH: 365 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 12Ser Gly Thr Ala Thr Tyr Ser Gly Asn Pro Ph #e Val Gly Val Thr Pro 1               5   #                10   #                15Trp Ala Asn Ala Tyr Tyr Ala Ser Glu Val Se #r Ser Leu Ala Ile Pro            20       #            25       #            30Ser Leu Thr Gly Ala Met Ala Thr Ala Ala Al #a Ala Val Ala Lys Val        35           #        40           #        45Pro Ser Phe Met Trp Leu Asp Thr Leu Asp Ly #s Thr Pro Leu Met Glu    50               #    55               #    60Gln Thr Leu Ala Asp Ile Arg Thr Ala Asn Ly #s Asn Gly Gly Asn Tyr65                   #70                   #75                   #80Ala Gly Gln Phe Val Val Ile Asp Leu Pro As #p Arg Asp Cys Ala Ala                85   #                90   #                95Leu Ala Ser Asn Gly Glu Tyr Ser Ile Ala As #p Gly Gly Val Ala Lys            100       #           105       #           110Tyr Lys Asn Tyr Ile Asp Thr Ile Arg Gln Il #e Val Val Glu Tyr Ser        115           #       120           #       125Asp Ile Arg Thr Leu Leu Val Ile Glu Pro As #p Ser Leu Ala Asn Leu    130               #   135               #   140Val Thr Asn Leu Gly Thr Pro Lys Cys Ala As #n Ala Gln Ser Ala Tyr145                 1 #50                 1 #55                 1 #60Leu Glu Cys Ile Asn Tyr Ala Val Thr Gln Le #u Asn Leu Pro Asn Val                165   #               170   #               175Ala Met Tyr Leu Asp Ala Gly His Ala Gly Tr #p Leu Gly Trp Pro Ala            180       #           185       #           190Asn Gln Asp Pro Ala Ala Gln Leu Phe Ala As #n Val Tyr Lys Asn Ala        195           #       200           #       205Ser Ser Pro Arg Ala Leu Arg Gly Leu Ala Th #r Asn Val Ala Asn Tyr    210               #   215               #   220Asn Gly Trp Asn Ile Thr Ser Pro Pro Ser Ty #r Thr Gln Gly Asn Ala225                 2 #30                 2 #35                 2 #40Val Tyr Asn Glu Lys Leu Tyr Ile His Ala Il #e Gly Pro Leu Leu Ala                245   #               250   #               255Asn His Gly Trp Ser Asn Ala Phe Phe Ile Th #r Asp Gln Gly Arg Ser            260       #           265       #           270Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp Gl #y Asp Trp Cys Asn Val        275           #       280           #       285Ile Gly Thr Gly Phe Gly Ile Arg Pro Ser Al #a Asn Thr Gly Asp Ser    290               #   295               #   300Leu Leu Asp Ser Phe Val Trp Val Lys Pro Gl #y Gly Glu Cys Asp Gly305                 3 #10                 3 #15                 3 #20Thr Ser Asp Ser Ser Ala Pro Arg Phe Asp Se #r His Cys Ala Leu Pro                325   #               330   #               335Asp Ala Leu Gln Pro Ala Pro Gln Ala Gly Al #a Trp Phe Gln Ala Tyr            340       #           345       #           350Phe Val Gln Leu Leu Thr Asn Ala Asn Pro Se #r Phe Leu        355           #       360           #       365<210> SEQ ID NO 13 <211> LENGTH: 1201 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(704) <221> NAME/KEY: CDS<222> LOCATION: (775)...(1201) <400> SEQUENCE: 13cag caa ccg ggt acc agc acc ccc gag gtc ca#t ccc aag ttg aca acc       48Gln Gln Pro Gly Thr Ser Thr Pro Glu Val Hi #s Pro Lys Leu Thr Thr 1               5   #                 10  #                 15tac aag tgt aca aag tcc ggg ggg tgc gtg gc#c cag gac acc tcg gtg       96Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val Al #a Gln Asp Thr Ser Val             20      #             25      #             30gtc ctt gac tgg aac tac cgc tgg atg cac ga#c gca aac tac aac tcg      144Val Leu Asp Trp Asn Tyr Arg Trp Met His As #p Ala Asn Tyr Asn Ser         35          #         40          #         45tgc acc gtc aac ggc ggc gtc aac acc acg ct#c tgc cct gac gag gcg      192Cys Thr Val Asn Gly Gly Val Asn Thr Thr Le #u Cys Pro Asp Glu Ala     50              #     55              #     60acc tgt ggc aag aac tgc ttc atc gag ggc gt#c gac tac gcc gcc tcg      240Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly Va #l Asp Tyr Ala Ala Ser 65                  # 70                  # 75                  # 80ggc gtc acg acc tcg ggc agc agc ctc acc at#g aac cag tac atg ccc      288Gly Val Thr Thr Ser Gly Ser Ser Leu Thr Me #t Asn Gln Tyr Met Pro                 85  #                 90  #                 95agc agc tct ggc ggc tac agc agc gtc tct cc#t cgg ctg tat ctc ctg      336Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser Pr #o Arg Leu Tyr Leu Leu            100       #           105       #           110gac tct gac ggt gag tac gtg atg ctg aag ct#c aac ggc cag gag ctg      384Asp Ser Asp Gly Glu Tyr Val Met Leu Lys Le #u Asn Gly Gln Glu Leu        115           #       120           #       125agc ttc gac gtc gac ctc tct gct ctg ccg tg#t gga gag aac ggc tcg      432Ser Phe Asp Val Asp Leu Ser Ala Leu Pro Cy #s Gly Glu Asn Gly Ser    130               #   135               #   140ctc tac ctg tct cag atg gac gag aac ggg gg#c gcc aac cag tat aac      480Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly Gl #y Ala Asn Gln Tyr Asn145                 1 #50                 1 #55                 1 #60acg gcc ggt gcc aac tac ggg agc ggc tac tg#c gat gct cag tgc ccc      528Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr Cy #s Asp Ala Gln Cys Pro                165   #               170   #               175gtc cag aca tgg agg aac ggc acc ctc aac ac#t agc cac cag ggc ttc      576Val Gln Thr Trp Arg Asn Gly Thr Leu Asn Th #r Ser His Gln Gly Phe            180       #           185       #           190tgc tgc aac gag atg gat atc ctg gag ggc aa#c tcg agg gcg aat gcc      624Cys Cys Asn Glu Met Asp Ile Leu Glu Gly As #n Ser Arg Ala Asn Ala        195           #       200           #       205ttg acc cct cac tct tgc acg gcc acg gcc tg#c gac tct gcc ggt tgc      672Leu Thr Pro His Ser Cys Thr Ala Thr Ala Cy #s Asp Ser Ala Gly Cys    210               #   215               #   220ggc ttc aac ccc tat ggc agc ggc tac aaa ag# gtgagcctga tgccactact     724Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys Se #r 225                 2#30                 2 #35acccctttcc tggcgctctc gcggttttcc atgctgacat ggttttccag c #tac tac     781                    #                  #                   #   Tyr Tyrggc ccc gga gat acc gtt gac acc tcc aag ac#c ttc acc atc atc acc      829Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Th #r Phe Thr Ile Ile Thr        240           #       245           #       250cag ttc aac acg gac aac ggc tcg ccc tcg gg#c aac ctt gtg agc atc      877Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gl #y Asn Leu Val Ser Ile    255               #   260               #   265acc cgc aag tac cag caa aac ggc gtc gac at#c ccc agc gcc cag ccc      925Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Il #e Pro Ser Ala Gln Pro270                 2 #75                 2 #80                 2 #85ggc ggc gac acc atc tcg tcc tgc ccg tcc gc#c tca gcc tac ggc ggc      973Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Al #a Ser Ala Tyr Gly Gly                290   #               295   #               300ctc gcc acc atg ggc aag gcc ctg agc agc gg#c atg gtg ctc gtg ttc     1021Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gl #y Met Val Leu Val Phe            305       #           310       #           315agc att tgg aac gac aac agc cag tac atg aa#c tgg ctc gac agc ggc     1069Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met As #n Trp Leu Asp Ser Gly        320           #       325           #       330aac gcc ggc ccc tgc agc agc acc gag ggc aa#c cca tcc aac atc ctg     1117Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly As #n Pro Ser Asn Ile Leu    335               #   340               #   345gcc aac aac ccc aac acg cac gtc gtc ttc tc#c aac atc cgc tgg gga     1165Ala Asn Asn Pro Asn Thr His Val Val Phe Se #r Asn Ile Arg Trp Gly350                 3 #55                 3 #60                 3 #65gac att ggg tct act acg aac tcg act gcg cc #c ccg                #     1201 Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala Pr #o Pro                370   #               375 <210> SEQ ID NO 14<211> LENGTH: 377 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 14Gln Gln Pro Gly Thr Ser Thr Pro Glu Val Hi #s Pro Lys Leu Thr Thr 1               5   #                10   #                15Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val Al #a Gln Asp Thr Ser Val            20       #            25       #            30Val Leu Asp Trp Asn Tyr Arg Trp Met His As #p Ala Asn Tyr Asn Ser        35           #        40           #        45Cys Thr Val Asn Gly Gly Val Asn Thr Thr Le #u Cys Pro Asp Glu Ala    50               #    55               #    60Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly Va #l Asp Tyr Ala Ala Ser65                   #70                   #75                   #80Gly Val Thr Thr Ser Gly Ser Ser Leu Thr Me #t Asn Gln Tyr Met Pro                85   #                90   #                95Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser Pr #o Arg Leu Tyr Leu Leu            100       #           105       #           110Asp Ser Asp Gly Glu Tyr Val Met Leu Lys Le #u Asn Gly Gln Glu Leu        115           #       120           #       125Ser Phe Asp Val Asp Leu Ser Ala Leu Pro Cy #s Gly Glu Asn Gly Ser    130               #   135               #   140Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly Gl #y Ala Asn Gln Tyr Asn145                 1 #50                 1 #55                 1 #60Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr Cy #s Asp Ala Gln Cys Pro                165   #               170   #               175Val Gln Thr Trp Arg Asn Gly Thr Leu Asn Th #r Ser His Gln Gly Phe            180       #           185       #           190Cys Cys Asn Glu Met Asp Ile Leu Glu Gly As #n Ser Arg Ala Asn Ala        195           #       200           #       205Leu Thr Pro His Ser Cys Thr Ala Thr Ala Cy #s Asp Ser Ala Gly Cys    210               #   215               #   220Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys Se #r Tyr Tyr Gly Pro Gly225                 2 #30                 2 #35                 2 #40Asp Thr Val Asp Thr Ser Lys Thr Phe Thr Il #e Ile Thr Gln Phe Asn                245   #               250   #               255Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu Va #l Ser Ile Thr Arg Lys            260       #           265       #           270Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser Al #a Gln Pro Gly Gly Asp        275           #       280           #       285Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala Ty #r Gly Gly Leu Ala Thr    290               #   295               #   300Met Gly Lys Ala Leu Ser Ser Gly Met Val Le #u Val Phe Ser Ile Trp305                 3 #10                 3 #15                 3 #20Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu As #p Ser Gly Asn Ala Gly                325   #               330   #               335Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser As #n Ile Leu Ala Asn Asn            340       #           345       #           350Pro Asn Thr His Val Val Phe Ser Asn Ile Ar #g Trp Gly Asp Ile Gly        355           #       360           #       365Ser Thr Thr Asn Ser Thr Ala Pro Pro     370               #   375<210> SEQ ID NO 15 <211> LENGTH: 1155 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(56) <221> NAME/KEY: CDS<222> LOCATION: (231)...(1155) <400> SEQUENCE: 15ggg gtc cga ttt gcc ggc gtt aac atc gcg gg#t ttt gac ttt ggc tgt       48Gly Val Arg Phe Ala Gly Val Asn Ile Ala Gl #y Phe Asp Phe Gly Cys 1               5   #                 10  #                 15acc aca ga gtgagtaccc ttgtttcctg gtgttgctgg ctggttgggc# gggtatacag    106 Thr Thr Aspcgaagcggac gcaagaacac cgccggtccg ccaccatcaa gatgtgggtg gt#aagcggcg    166gtgttttgta caactacctg acagctcact caggaaatga gaattaatgg aa#gtcttgtt    226 acag t ggc act tgc gtt acc tcg aag gtt tat# cct ccg ttg aag aac       273        Gly Thr Cys Val Thr Ser Ly#s Val Tyr Pro Pro Leu Lys Asn         20          #        25           #        30ttc acc ggc tca aac aac tac ccc gat ggc at#c ggc cag atg cag cac      321Phe Thr Gly Ser Asn Asn Tyr Pro Asp Gly Il #e Gly Gln Met Gln His     35              #     40              #     45ttc gtc aac gag gac ggg atg act att ttc cg#c tta cct gtc gga tgg      369Phe Val Asn Glu Asp Gly Met Thr Ile Phe Ar #g Leu Pro Val Gly Trp 50                  # 55                  # 60                  # 65cag tac ctc gtc aac aac aat ttg ggc ggc aa#t ctt gat tcc acg agc      417Gln Tyr Leu Val Asn Asn Asn Leu Gly Gly As #n Leu Asp Ser Thr Ser                 70  #                 75  #                 80att tcc aag tat gat cag ctt gtt cag ggg tg#c ctg tct ctg ggc gca      465Ile Ser Lys Tyr Asp Gln Leu Val Gln Gly Cy #s Leu Ser Leu Gly Ala             85      #             90      #             95tac tgc atc gtc gac atc cac aat tat gct cg#a tgg aac ggt ggg atc      513Tyr Cys Ile Val Asp Ile His Asn Tyr Ala Ar #g Trp Asn Gly Gly Ile        100           #       105           #       110att ggt cag ggc ggc cct act aat gct caa tt#c acg agc ctt tgg tcg      561Ile Gly Gln Gly Gly Pro Thr Asn Ala Gln Ph #e Thr Ser Leu Trp Ser    115               #   120               #   125cag ttg gca tca aag tac gca tct cag tcg ag#g gtg tgg ttc ggc atc      609Gln Leu Ala Ser Lys Tyr Ala Ser Gln Ser Ar #g Val Trp Phe Gly Ile130                 1 #35                 1 #40                 1 #45atg aat gag ccc cac gac gtg aac atc aac ac#c tgg gct gcc acg gtc      657Met Asn Glu Pro His Asp Val Asn Ile Asn Th #r Trp Ala Ala Thr Val                150   #               155   #               160caa gag gtt gta acc gca atc cgc aac gct gg#t gct acg tcg caa ttc      705Gln Glu Val Val Thr Ala Ile Arg Asn Ala Gl #y Ala Thr Ser Gln Phe            165       #           170       #           175atc tct ttg cct gga aat gat tgg caa tct gc#t ggg gct ttc ata tcc      753Ile Ser Leu Pro Gly Asn Asp Trp Gln Ser Al #a Gly Ala Phe Ile Ser        180           #       185           #       190gat ggc agt gca gcc gcc ctg tct caa gtc ac#g aac ccg gat ggg tca      801Asp Gly Ser Ala Ala Ala Leu Ser Gln Val Th #r Asn Pro Asp Gly Ser    195               #   200               #   205aca acg aat ctg att ttt gac gtg cac aaa ta#c ttg gac tca gac aac      849Thr Thr Asn Leu Ile Phe Asp Val His Lys Ty #r Leu Asp Ser Asp Asn210                 2 #15                 2 #20                 2 #25tcc ggt act cac gcc gaa tgt act aca aat aa#c att gac ggc gcc ttt      897Ser Gly Thr His Ala Glu Cys Thr Thr Asn As #n Ile Asp Gly Ala Phe                230   #               235   #               240tct ccg ctt gcc act tgg ctc cga cag aac aa#t cgc cag gct atc ctg      945Ser Pro Leu Ala Thr Trp Leu Arg Gln Asn As #n Arg Gln Ala Ile Leu            245       #           250       #           255aca gaa acc ggt ggt ggc aac gtt cag tcc tg#c ata caa gac atg tgc      993Thr Glu Thr Gly Gly Gly Asn Val Gln Ser Cy #s Ile Gln Asp Met Cys        260           #       265           #       270cag caa atc caa tat ctc aac cag aac tca ga#t gtc tat ctt ggc tat     1041Gln Gln Ile Gln Tyr Leu Asn Gln Asn Ser As #p Val Tyr Leu Gly Tyr    275               #   280               #   285gtt ggt tgg ggt gcc gga tca ttt gat agc ac#g tat gtc ctg acg gaa     1089Val Gly Trp Gly Ala Gly Ser Phe Asp Ser Th #r Tyr Val Leu Thr Glu290                 2 #95                 3 #00                 3 #05aca ccg act agc agt ggt aac tca tgg acg ga#c aca tcc ttg gtc agc     1137Thr Pro Thr Ser Ser Gly Asn Ser Trp Thr As #p Thr Ser Leu Val Ser                310   #               315   #               320tcg tgt ctc gca aga aag          #                   #                  #1155 Ser Cys Leu Ala Arg Lys             325 <210> SEQ ID NO 16<211> LENGTH: 327 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 16Gly Val Arg Phe Ala Gly Val Asn Ile Ala Gl #y Phe Asp Phe Gly Cys 1               5   #                10   #                15Thr Thr Asp Gly Thr Cys Val Thr Ser Lys Va #l Tyr Pro Pro Leu Lys            20       #            25       #            30Asn Phe Thr Gly Ser Asn Asn Tyr Pro Asp Gl #y Ile Gly Gln Met Gln        35           #        40           #        45His Phe Val Asn Glu Asp Gly Met Thr Ile Ph #e Arg Leu Pro Val Gly    50               #    55               #    60Trp Gln Tyr Leu Val Asn Asn Asn Leu Gly Gl #y Asn Leu Asp Ser Thr65                   #70                   #75                   #80Ser Ile Ser Lys Tyr Asp Gln Leu Val Gln Gl #y Cys Leu Ser Leu Gly                85   #                90   #                95Ala Tyr Cys Ile Val Asp Ile His Asn Tyr Al #a Arg Trp Asn Gly Gly            100       #           105       #           110Ile Ile Gly Gln Gly Gly Pro Thr Asn Ala Gl #n Phe Thr Ser Leu Trp        115           #       120           #       125Ser Gln Leu Ala Ser Lys Tyr Ala Ser Gln Se #r Arg Val Trp Phe Gly    130               #   135               #   140Ile Met Asn Glu Pro His Asp Val Asn Ile As #n Thr Trp Ala Ala Thr145                 1 #50                 1 #55                 1 #60Val Gln Glu Val Val Thr Ala Ile Arg Asn Al #a Gly Ala Thr Ser Gln                165   #               170   #               175Phe Ile Ser Leu Pro Gly Asn Asp Trp Gln Se #r Ala Gly Ala Phe Ile            180       #           185       #           190Ser Asp Gly Ser Ala Ala Ala Leu Ser Gln Va #l Thr Asn Pro Asp Gly        195           #       200           #       205Ser Thr Thr Asn Leu Ile Phe Asp Val His Ly #s Tyr Leu Asp Ser Asp    210               #   215               #   220Asn Ser Gly Thr His Ala Glu Cys Thr Thr As #n Asn Ile Asp Gly Ala225                 2 #30                 2 #35                 2 #40Phe Ser Pro Leu Ala Thr Trp Leu Arg Gln As #n Asn Arg Gln Ala Ile                245   #               250   #               255Leu Thr Glu Thr Gly Gly Gly Asn Val Gln Se #r Cys Ile Gln Asp Met            260       #           265       #           270Cys Gln Gln Ile Gln Tyr Leu Asn Gln Asn Se #r Asp Val Tyr Leu Gly        275           #       280           #       285Tyr Val Gly Trp Gly Ala Gly Ser Phe Asp Se #r Thr Tyr Val Leu Thr    290               #   295               #   300Glu Thr Pro Thr Ser Ser Gly Asn Ser Trp Th #r Asp Thr Ser Leu Val305                 3 #10                 3 #15                 3 #20Ser Ser Cys Leu Ala Arg Lys                 325 <210> SEQ ID NO 17<211> LENGTH: 72 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(72) <400> SEQUENCE: 17cgt ggc acc acc acc acc cgc cgc cca gcc ac#t acc act gga agc tct       48Arg Gly Thr Thr Thr Thr Arg Arg Pro Ala Th #r Thr Thr Gly Ser Ser 1               5   #                 10  #                 15ccc gga cct acc cag tct cac tac      #                  #                72 Pro Gly Pro Thr Gln Ser His Tyr              20<210> SEQ ID NO 18 <211> LENGTH: 24 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 18Arg Gly Thr Thr Thr Thr Arg Arg Pro Ala Th #r Thr Thr Gly Ser Ser 1               5   #                10   #                15Pro Gly Pro Thr Gln Ser His Tyr             20 <210> SEQ ID NO 19<211> LENGTH: 129 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(129) <400> SEQUENCE: 19ggc gct gca agc tca agc tcg tcc acg cgc gc#c gcg tcg acg act tct       48Gly Ala Ala Ser Ser Ser Ser Ser Thr Arg Al #a Ala Ser Thr Thr Ser 1               5   #                 10  #                 15cga gta tcc ccc aca aca tcc cgg tcg agc tc#c gcg acg cct cca cct       96Arg Val Ser Pro Thr Thr Ser Arg Ser Ser Se #r Ala Thr Pro Pro Pro             20      #             25      #             30ggt tct act act acc aga gta cct cca gtc gg #a                  #        129 Gly Ser Thr Thr Thr Arg Val Pro Pro Val Gl #y         35          #         40 <210> SEQ ID NO 20 <211> LENGTH: 43<212> TYPE: PRT <213> ORGANISM: Trichoderma longibrachiatum<400> SEQUENCE: 20 Gly Ala Ala Ser Ser Ser Ser Ser Thr Arg Al#a Ala Ser Thr Thr Ser  1               5   #                10  #                15 Arg Val Ser Pro Thr Thr Ser Arg Ser Ser Se#r Ala Thr Pro Pro Pro             20       #            25      #            30 Gly Ser Thr Thr Thr Arg Val Pro Pro Val Gl #y        35           #        40 <210> SEQ ID NO 21 <211> LENGTH: 81<212> TYPE: DNA <213> ORGANISM: Trichoderma longibrachiatum<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(81)<400> SEQUENCE: 21 ccc ccg cct gcg tcc agc acg acg ttt tcg ac#t aca ccg agg agc tcg       48Pro Pro Pro Ala Ser Ser Thr Thr Phe Ser Th #r Thr Pro Arg Ser Ser 1               5   #                 10  #                 15acg act tcg agc agc ccg agc tgc acg cag ac #t                  #         81 Thr Thr Ser Ser Ser Pro Ser Cys Thr Gln Th #r             20      #             25 <210> SEQ ID NO 22<211> LENGTH: 27 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 22Pro Pro Pro Ala Ser Ser Thr Thr Phe Ser Th #r Thr Pro Arg Ser Ser 1               5   #                10   #                15Thr Thr Ser Ser Ser Pro Ser Cys Thr Gln Th #r             20      #            25 <210> SEQ ID NO 23 <211> LENGTH: 102 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(102) <400> SEQUENCE: 23ccg gga gcc act act atc acc act tcg acc cg#g cca cca tcc ggt cca       48Pro Gly Ala Thr Thr Ile Thr Thr Ser Thr Ar #g Pro Pro Ser Gly Pro 1               5   #                 10  #                 15acc acc acc acc agg gct acc tca aca agc tc#a tca act cca ccc acg       96Thr Thr Thr Thr Arg Ala Thr Ser Thr Ser Se #r Ser Thr Pro Pro Thr             20      #             25      #             30agc tct                 #                   #                  #          102 Ser Ser <210> SEQ ID NO 24 <211> LENGTH: 34<212> TYPE: PRT <213> ORGANISM: Trichoderma longibrachiatum<400> SEQUENCE: 24 Pro Gly Ala Thr Thr Ile Thr Thr Ser Thr Ar#g Pro Pro Ser Gly Pro  1               5   #                10  #                15 Thr Thr Thr Thr Arg Ala Thr Ser Thr Ser Se#r Ser Thr Pro Pro Thr             20       #            25      #            30 Ser Ser <210> SEQ ID NO 25 <211> LENGTH: 51<212> TYPE: DNA <213> ORGANISM: Trichoderma longibrachiatum<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(51)<400> SEQUENCE: 25 atg tat cgg aag ttg gcc gtc atc tcg gcc tt#c ttg gcc aca gct cgt       48Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Ph #e Leu Ala Thr Ala Arg 1               5   #                 10  #                 15gct                   #                   #                  #             51 Ala <210> SEQ ID NO 26 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 26Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Ph #e Leu Ala Thr Ala Arg 1               5   #                10   #                15 Ala<210> SEQ ID NO 27 <211> LENGTH: 72 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(72) <400> SEQUENCE: 27atg att gtc ggc att ctc acc acg ctg gct ac#g ctg gcc aca ctc gca       48Met Ile Val Gly Ile Leu Thr Thr Leu Ala Th #r Leu Ala Thr Leu Ala 1               5   #                 10  #                 15gct agt gtg cct cta gag gag cgg      #                  #                72 Ala Ser Val Pro Leu Glu Glu Arg              20<210> SEQ ID NO 28 <211> LENGTH: 24 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 28Met Ile Val Gly Ile Leu Thr Thr Leu Ala Th #r Leu Ala Thr Leu Ala 1               5   #                10   #                15Ala Ser Val Pro Leu Glu Glu Arg             20 <210> SEQ ID NO 29<211> LENGTH: 66 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(66) <400> SEQUENCE: 29atg gcg ccc tca gtt aca ctg ccg ttg acc ac#g gcc atc ctg gcc att       48Met Ala Pro Ser Val Thr Leu Pro Leu Thr Th #r Ala Ile Leu Ala Ile 1               5   #                 10  #                 15gcc cgg ctc gtc gcc gcc          #                   #                  #  66 Ala Arg Leu Val Ala Ala              20 <210> SEQ ID NO 30<211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 30Met Ala Pro Ser Val Thr Leu Pro Leu Thr Th #r Ala Ile Leu Ala Ile 1               5   #                10   #                15Ala Arg Leu Val Ala Ala             20 <210> SEQ ID NO 31<211> LENGTH: 63 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(63) <400> SEQUENCE: 31atg aac aag tcc gtg gct cca ttg ctg ctt gc#a gcg tcc ata cta tat       48Met Asn Lys Ser Val Ala Pro Leu Leu Leu Al #a Ala Ser Ile Leu Tyr 1               5   #                 10  #                 15ggc ggc gcc gtc gca            #                   #                  #    63 Gly Gly Ala Val Ala              20 <210> SEQ ID NO 32<211> LENGTH: 21 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 32Met Asn Lys Ser Val Ala Pro Leu Leu Leu Al #a Ala Ser Ile Leu Tyr 1               5   #                10   #                15Gly Gly Ala Val Ala             20 <210> SEQ ID NO 33 <211> LENGTH: 777<212> TYPE: DNA <213> ORGANISM: Trichoderma longibrachiatum<400> SEQUENCE: 33aaaccagctg tgaccagtgg gcaaccttca ctggcaacgg ctacacagtc ag#caacaacc     60tttggggagc atcagccggc tctggatttg gctgcgtgac ggcggtatcg ct#cagcggcg    120gggcctcctg gcacgcagac tggcagtggt ccggcggcca gaacaacgtc aa#gtcgtacc    180agaactctca gattgccatt ccccagaaga ggaccgtcaa cagcatcagc ag#catgccca    240ccactgccag ctggagctac agcgggagca acatccgcgc taatgttgcg ta#tgacttgt    300tcaccgcagc caacccgaat catgtcacgt actcgggaga ctacgaactc at#gatctggt    360aagccataag aagtgaccct ccttgatagt ttcgactaac aacatgtctt ga#ggcttggc    420aaatacggcg atattgggcc gattgggtcc tcacagggaa cagtcaacgt cg#gtggccag    480agctggacgc tctactatgg ctacaacgga gccatgcaag tctattcctt tg#tggcccag    540accaacacta ccaactacag cggagatgtc aagaacttct tcaattatct cc#gagacaat    600aaaggataca acgctgcagg ccaatatgtt cttagtaagt caccctcact gt#gactgggc    660tgagtttgtt gcaacgtttg ctaacaaaac cttcgtatag gctaccaatt tg#gtaccgag    720cccttcacgg gcagtggaac tctgaacgtc gcatcctgga ccgcatctat ca#actaa       777 <210> SEQ ID NO 34 <211> LENGTH: 218 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 34Gln Thr Ser Cys Asp Gln Trp Ala Thr Phe Th #r Gly Asn Gly Tyr Thr 1               5   #                10   #                15Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gl #y Ser Gly Phe Gly Cys            20       #            25       #            30Val Thr Ala Val Ser Leu Ser Gly Gly Ala Se #r Trp His Ala Asp Trp        35           #        40           #        45Gln Trp Ser Gly Gly Gln Asn Asn Val Lys Se #r Tyr Gln Asn Ser Gln    50               #    55               #    60Ile Ala Ile Pro Gln Lys Arg Thr Val Asn Se #r Ile Ser Ser Met Pro65                   #70                   #75                   #80Thr Thr Ala Ser Trp Ser Tyr Ser Gly Ser As #n Ile Arg Ala Asn Val                85   #                90   #                95Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro As #n His Val Thr Tyr Ser            100       #           105       #           110Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Ly #s Tyr Gly Asp Ile Gly        115           #       120           #       125Pro Ile Gly Ser Ser Gln Gly Thr Val Asn Va #l Gly Gly Gln Ser Trp    130               #   135               #   140Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gl #n Val Tyr Ser Phe Val145                 1 #50                 1 #55                 1 #60Ala Gln Thr Asn Thr Thr Asn Tyr Ser Gly As #p Val Lys Asn Phe Phe                165   #               170   #               175Asn Tyr Leu Arg Asp Asn Lys Gly Tyr Asn Al #a Ala Gly Gln Tyr Val            180       #           185       #           190Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe Th #r Gly Ser Gly Thr Leu        195           #       200           #       205Asn Val Ala Ser Trp Thr Ala Ser Ile Asn     210               #   215<210> SEQ ID NO 35 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 35atgaagttcc ttcaagtcct ccctgccctc ataccggccg ccctggcc  #                48 <210> SEQ ID NO 36 <211> LENGTH: 16 <212> TYPE: PRT<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 36Met Lys Phe Leu Gln Val Leu Pro Ala Leu Il #e Pro Ala Ala Leu Ala 1               5   #                10   #                15<210> SEQ ID NO 37 <211> LENGTH: 57 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 37agctcgtaga gcgttgactt gcctgtggtc tgtccagacg ggggacgata ga#atgcg        57 <210> SEQ ID NO 38 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 38gtcaccttct ccaacatcaa gttcggaccc attggcagca ccggctaa  #                48 <210> SEQ ID NO 39 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 39ggggtttaaa cccgcgggga tt            #                  #                 22 <210> SEQ ID NO 40 <211> LENGTH: 15 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 40tgagccgagg cctcc               #                   #                  #    15 <210> SEQ ID NO 41 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Trichoderma longibrachiatum <400> SEQUENCE: 41agcttgagat ctgaagct              #                   #                  #  18 <210> SEQ ID NO 42 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic oligonucleotide <400> SEQUENCE: 42ctagaggagc ggtcgggaac cgctac           #                  #              26 <210> SEQ ID NO 43 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic peptide based o #n oligonucleotide      (SEQ ID NO:42) <400> SEQUENCE: 43Leu Glu Glu Arg Ser Gly Thr Ala Thr  1               5

We claim:
 1. A method of treating cellulose containing fabrics withcellulase comprising the steps of: (a) contacting said cellulosecontaining fabric with a treating composition comprising an effectiveamount of a naturally occurring cellulase which lacks cellulose bindingdomain and which exhibits endoglucanase activity; and (b) incubatingsaid cellulose containing fabric in contact with said cellulose underconditions effective to treat said fabric.
 2. The method according toclaim 1, wherein said cellulose containing fabric comprises a cottoncontaining fabric.
 3. The method according to claim 2, wherein saidcotton containing fabric comprises dyed denim.
 4. The method accordingto claim 1, wherein said treating composition comprises a naturallyoccurring cellulase which lacks a cellulose binding domain and whichexhibits endoglucanase activity, said cellulase being present in aconcentration of about 0.1 to about 1,000 ppm total protein.
 5. Themethod according to claim 1, wherein said treating composition comprisesa naturally occurring cellulase which lacks a cellulose binding domainand which exhibits endoglucanase activity, said cellulase being presentin a concentration of about 0.2 to about 500 ppm.
 6. The methodaccording to claim 1, wherein said naturally occurring cellulose whichlacks a cellulose binding domain which exhibits endoglucanase activityis derived from a microorganism which is a fungus or bacteria.
 7. Themethod according to claim 6, wherein said fungus is Trichoderma sp. 8.The method according to claim 7, wherein said Trichoderma sp. isTrichoderma longibrachiatum.
 9. The method according to claim 1, whereinsaid treating method comprises stonewashing the cellulose containingfabric and said treating composition comprises a stonewashingcomposition.
 10. The method according to claim 9, wherein said cellulosecontaining fabric is colored.
 11. The method according to claim 10,wherein said colored fabric is dyed denim.
 12. The method according toclaim 9, comprising the additional step of treating said fabric withpumice simultaneously with, before or after said treating step.
 13. Themethod according to claim 9, wherein said stonewashing compositioncomprises a naturally occurring cellulase which lacks a cellulosebinding domain and which exhibits endoglucanase activity.
 14. The methodaccording to claim 13, wherein said naturally occurring cellulase whichlacks a cellulose binding domain and which exhibits endoglucanaseactivity is EGIII.
 15. The method according to claim 13, wherein saidnaturally occurring cellulase which lacks a cellulose binding domain andwhich exhibits endoglucanase activity, said cellulase being present in aconcentration of about 10 to about 400 ppm total protein.
 16. The methodaccording to claim 13, wherein said naturally occurring cellulase whichlacks a cellulose binding domain and which exhibits endoglucanaseactivity, said cellulase being present in a concentration of about 20 toabout 100 ppm total protein.
 17. The method according to claim 1,wherein said treating method comprises washing the cellulose containingfabric and said treating composition is a detergent compositioncomprising a surfactant.
 18. The method according to claim 17, whereinsaid surfactant comprises nonionic ethoxylated alkyl phenols nonionicethoxylated alcohols.
 19. The method according to claim 17, wherein saidtreating composition comprises a naturally occurring cellulase whichlacks a cellulose binding domain and which exhibits endoglucanaseactivity.
 20. The method according to claim 19, wherein said naturallyoccurring cellulase which lacks a cellulose binding domain and whichexhibits endoglucanase activity, said cellulase being present in aconcentration of about 0.1 to about 1000 ppm.
 21. The method accordingto claim 19, wherein said naturally occurring cellulase which lacks acellulose binding domain and which exhibits endoglucanase activity, saidcellulose being present in a concentration of about 0.2 to about 500ppm.
 22. A composition for treating a cellulose containing fabriccomprising a naturally occurring cellulase which lacks a cellulosebinding domain and which exhibits endoglucanase activity.
 23. Thecomposition according to claim 22, wherein said composition is usefulfor stonewashing cellulose containing fabric and further contains asurfactant.
 24. The composition according to claim 23, wherein saidnaturally occurring cellulase which lacks a cellulose binding domain andwhich exhibits endoglucanase activity, said cellulase being present in aconcentration of about 1 to about 1000 ppm total protein.
 25. Thecomposition according to claim 22, wherein said composition is useful asa detergent and further comprises a surfactant.
 26. The compositionaccording to claim 25, wherein said surfactant comprises nonionicethoxylated alkyl phenols or nonionic ethoxylated alcohols.
 27. Thecomposition according to claim 25, wherein said naturally occurringcellulase which lacks a cellulose binding domain and which exhibitsendoglucanase activity, said cellulase being present in a concentrationof about 0.1 to about 1000 ppm total protein.
 28. The compositionaccording to claim 22, wherein said treating composition is formulatedas a pre-soak.
 29. A fabric produced by the process of claim
 1. 30. Afabric produced by the process of claim
 9. 31. A fabric produced by theprocess of claim
 17. 32. The fabric of claim 29, wherein said fabriccomprises dyed denim.